HK1116998A - Soy compositions having improved organoleptic properties and methods of generation - Google Patents

Description

Soy compositions having improved organoleptic properties and methods of production

Background

The present invention claims priority from U.S. provisional patent application serial No. 60/521,846, filed on 9/7/2004, the entire disclosure of which is expressly incorporated herein by reference.

1. Field of the invention

The present invention relates to the field of nutrition and food science. In particular, the present invention relates to soy compositions having improved organoleptic properties, such as reduced odor, and methods of using and producing the same.

2. Description of the related Art

Soybean provides high quality proteins beneficial to human health (Hermansen et al, 2003; Bazzano et al, 2001; U.S. food and drug administration, 1999). Over The past three decades, The demand for soybeans to make soy foods has risen less than expected (Wolfe and Cowan, 1975; SoySource, The United Soyboard Board 1999). This is due, in part, to the undesirable odor associated with soy products (McLeod and Ames, 1988; Freese, 1999). This undesirable soy odor is commonly described as "beany flavor". Ingredients that impart beany flavour characteristics to soybeans include a number of volatile fatty acids, aliphatic carbonyl compounds, amines, alcohols, aldehydes and furans, which derive from the action of enzymes on the various compounds present in soybeans and the further oxidation of these compounds by a number of mechanisms (Wolfe and Cowan, 1975; Sessa and Rackis, 1977).

Kobay Ashi et al (1995) concluded that the main substances responsible for the odor of uncooked soymilk were (trans ) -2, 4-nonadienal, (trans ) -2, 4-decadienal, hexanal, 2-pentylfuran, 1-octen-3-one, (trans) -2-nonenal and (trans, cis) -2, 4-nonadienal. The most intense odors extracted from heat-treated soymilk were identified as (trans ) -2, 4-decadienal and n-hexanal (Feng, doctrine at the university of kannel, 2000). The formation of (trans ) -2, 4-decadienal occurs slowly at room temperature (Frankel, 1988), but this reaction is enhanced by thermal degradation under the thermal conditions during soybean processing (Lin, 2003). Other odor causing substances are (trans) -4, 5-epoxy- (E) -2-decenal (formed from 2, 4-decadienal), (trans, cis) -2, 6-nonadienal, (trans) -2-nonenal, (trans ) -2, 4-nonadienal, maltol, vanillin and β -damascenone. The most powerful odorants in soymilk, determined by odor assay detection of the minimum headspace volume required, are hexanal, acetaldehyde, methyl mercaptan, dimethyl trisulfide and 2-pentylfuran (Boatright, 2002).

The most powerful odorants of soy protein isolates were identified as dimethyltrithiol, (trans ) -2, 4-decadienal, 2-pentylpyridine, (trans ) -2, 4-nonadienal, hexanal, acetophenone and 1-octen-3-one (Boatright and Lei, 1999). The mechanism of formation of methyl mercaptan and dimethyl trisulfide involves free radicals formed by lipid oxidation (Lei and Boatright, 2003) and products of enzymes such as cysteine synthase (Boatright, 2003, poster 45C-26, IFT annual meeting, Chicago).

The formation of 2-pentylpyridine occurs from a spontaneous reaction of 2, 4-decadienal and ammonia at room temperature. Arginine, lysine, asparagine, and glutamine, which are free amino acids, may increase the formation of 2-pentylpyridine by providing ammonia during processing of soy protein. (Zhou and Boatright, 2000; Kim et al, 1996). Free amino acids can also form other undesirable products. Exposure of asparagine and glucose to high temperatures results in the formation of acrylamide (Jung et al, 2003). Arginine exposed to cooking temperatures can form mutagens (Knize et al, 1994). Free arginine is abundant in soybeans that lack β -conglycinin and glycinin (Takahashi et al, 2003).

Once formed, odors are difficult to remove from the soy component because they bind to proteins (Franzen and Kinsella, 1974). The quality of natural flavors added to soy foods is also adversely affected due to the incorporation of some odor onto the soy protein. Carbonyl compounds and 2-pentylpyridine bind to the glycinin fraction with greater affinity than to the β -conglycinin fraction (Zhou et al, 2002; O' Keefe et al, 1991). Extraction of oil body binding proteins and polar lipids can significantly reduce the amount of odor associated with soy protein isolate (Samoto et al, 1998).

Texture due to protein-protein interactions has a greater effect on odor intensity than nasal (in-nose) odor concentration (Weel et al, 2002). Soy protein can contribute to poor organoleptic quality of soy beverages by forming insoluble aggregates and lime mouthfeel (chalky mouthfeel). Among the major soy proteins, glycinin is responsible for pH and Ca⁺²The resulting insolubilization is more sensitive (Yuan, 2002), so soybeans with a low ratio of glycinin to beta-conglycinin are useful for producing soluble soy proteinAre useful (U.S. Pat. No.6,171,640). Lipid oxidation reactions also affect protein solubility. Antioxidants may be added during the soy protein isolate manufacturing process to limit free radical induced protein oxidation and increase the yield of soluble protein (U.S. Pat. No. 5,777,080). Some peptides can react with polysaccharides during processing to form antioxidant compounds (Matsumura, 2003).

Color affects freshness and taste (Joshi, 2000). Small amounts of reducing sugars and aldehydes formed by lipid oxidation undergo Maillard browning reactions with the amino groups of proteins upon heating to form brown pigments (Kwok et al, 1999). Soymilk with higher aldehyde content will produce darker, less appealing colors after thermal processing. On the other hand, lipid oxidation during soymilk processing discolors the yellow pigments of soymilk (Obata and Matsuura, 1997).

Soybeans may be refined to improve flavor by extracting lipids and other ingredients by alcohol extraction, enzyme treatment, protein clot (protein cure) washing, protein ultrafiltration, and/or the application of flash evaporation. These processes increase the cost of the soy protein component and generally reduce the amount of health-beneficial bioavailable components (e.g., fiber, oligosaccharides, isoflavones, polyunsaturated fatty acids, tocopherols, phospholipids, bioactive peptides). Processing methods to improve the organoleptic properties of soy protein ingredients are limited in their effectiveness due to the incorporation of odor onto the soy protein and due to the presence of conditions (pH8-10) that promote the formation of odor. Soybeans lacking one to three of lipoxygenases (lipoxygenases) 1, 2 and 3 were constructed using mutagenic breeding to reduce the formation of beany odors (Hajika et al, 1991). Analysis of the aroma of soymilk and soybean meal made from soybeans lacking the three lipoxygenases revealed several lower odor levels, but higher levels of 1-octen-3-ol, compared to parental soybean lines containing all three lipoxygenases (Hao et al, 2002). Similar levels of 2, 4-decadienal were found in defatted soy flour and soy protein isolate made from one soybean line lacking three lipoxygenases and two other soybean lines (Boatright et al, 1998). Soy foods prepared from soybeans lacking lipoxygenase had improved flavor compared to foods prepared from control soybeans (Wilson, 1996). Soymilk prepared from soybeans lacking three lipoxygenases was perceived as bitter compared to control soybeans, particularly after 15 months of seed storage, but this difference was expected to be eliminated by the addition of sugar (Torres-Penaranda and Reitmeirer, 2001).

It has been proposed to make transgenic modifications to improve the flavor of soybean by reducing the levels of polyunsaturated fatty acids (U.S. patent No. 5,981,781), lipoxygenases (U.S. patent application No. 20030074693) and/or hydroperoxide lyases (U.S. patent No.6,444,874). Soybeans containing less than 10% polyunsaturated fatty acids and more than 75% oleic acid produce frying oils that are less palatable than those containing higher levels of polyunsaturated fatty acids (Warner et al, 2001).

Chemicals such as polyphosphates (U.S. patent No.6,355,296) can be used to limit off-flavor generation and improve protein solubility. Other additives such as gallic acid (PCT WO 01/06866) or aldehyde oxidase (Maheshwari et al, 1997) can be used to remove odors.

Little published information is available regarding the effect of natural genetic variation on the flavor and color quality of soybeans. Thiobarbituric acid values of 16 soybean varieties were determined and no correlation with soybean vitamin E content was found as a measure of lipid oxidation (Dahuja and Madaan, 2004). The amounts of 2-pentylpyridine and 2, 4-decadienal in soybean meal and soybean protein isolates prepared from three soybean varieties were determined (Zhou and Boatright, 1999). The effect of drying conditions on the removal of chlorophyll, a green pigment, from soybeans was investigated (Salete et al, 2003; Sinnecker et al, 2002).

Scientists have demonstrated over the past decade that oils made from soybeans lacking lipoxygenase do not have improved oxidative stability. The soy protein produced from lipoxygenase-free soybeans still contains significant levels of beany flavor (Maheshwari et al, 1997).

The first step in preparing soymilk or soy protein ingredients is to dehull (or dehull) the soybeans to produce soybean pulp. Hypocotyls can also be separated from cotyledons. Soybean pulp is defined as dehulled soybeans, which may or may not include cotyledons. A method of preparing soybean pulp is described in, for example, U.S. patent No. 5,727,689. One method of dehulling includes, but is not limited to, passing the seeds between counter-current rollers or crushing rollers and sucking the light hulls away to leave the pulp. The pulp can be soaked in water to produce soymilk, or the pulp can be flaked and extracted with hexane as an initial step to prepare defatted soy flour, soy protein concentrate, soy protein isolate, and purified protein fractions as desired.

The present invention provides a novel method to determine the ability of soybean pulp to prevent the production of the major odor compounds identified as 2, 4-decadienal, hexanal, hexanol, and 1-octen-3-ol. These compounds were selected as an indicator of the extent of the different types of oxidation reactions. Hexanal and hexanol are produced by the decomposition of hydroperoxide containing compounds (peroxides at the 9 and 12 carbon positions of fatty acids) by hydroperoxide lyase and alcohol dehydrogenase. 2, 4-decadienal is a breakdown product of the lipoxygenase pathway which is not known to be involved in hydroperoxide lyase. 1-octen-3-ol is formed by the action of a hydroperoxide lyase on the hydroperoxide formed at the 10-carbon position of linoleic acid. These compounds may react further by additional processing to form a more intense odor. For example, 2, 4-decadienal is involved in the formation of 2-pentylpyridine and 1-octen-3-ol is involved in the formation of 1-octen-3-one.

Summary of The Invention

In one aspect, the present invention provides a soybean pulp composition produced from soybeans comprising lipoxygenases 1, 2 and 3, wherein said composition comprises greater than 10% linoleic acid as a percentage of total fatty acids and comprises less than 20 μ g/g of total 2, 4-decadienal plus hexanal plus hexanol after oxidation under mild aqueous conditions. The composition may or may not comprise the individual lipoxygenases or any combination thereof, and may comprise an inactivated lipoxygenase. In one embodiment, the composition comprises lipoxygenase-2. In certain embodiments, the compositions provided herein can comprise less than about 15 μ g or less than about 18 μ g of total 2, 4-decadienal plus hexanal plus hexanol. In other embodiments, the composition may comprise from about 6 μ g to about 20 μ g, from about 10 μ g to 20 μ g, or from about 12 to 18 μ g of total 2, 4-decadienal plus hexanal plus hexanol. In other embodiments, the composition may comprise less than 4% linolenic acid as a percentage of total fatty acids, including less than about 3% and about 1% to 4% or about 2% to about 4%.

In another embodiment, the compositions of the present invention may comprise less than 2000 μ g/gram of free arginine and/or less than 400 μ g/gram of free asparagine, including less than about 1800 μ g/gram of free arginine and/or less than about 350 μ g/gram of free asparagine. Such compositions may comprise in certain embodiments about 300. mu.g to about 2000. mu.g/g of free arginine, including about 500-. The compositions provided herein may also comprise, in some embodiments, from about 50 μ g to about 400 μ g/g of free asparagine, including about 100-.

In yet another embodiment, the compositions provided herein can have a color of: according to wherein L^*Represents lightness and b^*CIE-L representing the hue on the blue (-) -yellow (+) color axis^*a^*b^*The system is monitored and measured as b^*A value less than 30 and L^*The value is greater than 80. In certain embodiments, the compositions of the present invention may have a b-measure^*Colors having values of about 18 to 30, about 20 to 30, about 25 to 30, and less than about 25. In other embodiments, the compositions provided herein can have an L of about 80 to 100, about 80 to 90, and greater than about 90^*The value is obtained. In certain embodiments, the compositions provided herein can comprise a 1-octen-3-ol content of less than 8 μ g/g after oxidation under mild aqueous conditions, including less than about 6 μ g, less than about 5 μ g, from about 1.3 to about 8 μ g, from about 2 to about 8 μ g, and from about 4 to about 8 μ g. The compositions provided herein may also have a viscosity of greater than30% protein beta-conglycinin, with less than 25% protein glycinin. Such compositions may be further defined as having greater than about 40% of the protein being beta-conglycinin, and having from about 30% to about 60%, from about 40% to about 60%, from about 35% to about 55%, and from about 30% to about 50% of the protein being beta-conglycinin. Such compositions may be further defined as having glycinin contents of less than about 20%, 15% and 10%, and glycinin which may comprise about 0% -25%, 5% -20%, 1% -25% and about 10-25% protein.

In another aspect of the invention, a soy pulp composition is provided having greater than 30% of the protein as beta-conglycinin, less than 25% of the protein as glycinin, less than 5,000 μ g/g free arginine, and less than 900 μ g/g free asparagine. Such compositions may comprise, in certain embodiments, about 300-5,000. mu.g/g, about 1,000-5,000. mu.g/g, about 3,000-5,000. mu.g/g, about 1,000-4,000. mu.g/g, and about 500-2,000. mu.g/g of free arginine. Such compositions may in certain embodiments comprise less than 400. mu.g/g of free asparagine, from about 50 to about 400. mu.g/g, from about 100-700. mu.g/g and from about 200-900. mu.g/g of free asparagine. In one embodiment, the composition has less than 2,000 μ g/gram of free arginine and less than 400 μ g/gram of free asparagine.

In another embodiment, provided compositions comprise a 1-octen-3-ol content of less than 4 μ g/g after oxidation under mild aqueous conditions, including less than about 3 μ g, about 1.3 μ g to 4 μ g, and about 2 μ g to 4 μ g/g. In still other embodiments, the linolenic acid concentration of the composition is between 1% and 14% of the total fatty acids, including about 3-14%, about 5-14%, about 1.5% -12%, about 3-12%, and about 7-14%. In yet another embodiment, the linoleic acid concentration of the composition is between 10% and 60% of the total fatty acids, including between about 10% and 50%, between about 10% and 40%, between about 15% and 60%, between about 20% and 50%, and between about 20% and 60%.

In certain embodiments, the invention provides compositions comprising a peptide of formula (I)A legume pulp composition may be defined as lacking one or more lipoxygenases. In one embodiment, the soy pulp composition provided herein can be defined as lacking lipoxygenase-2. In other embodiments, any combination of lipoxygenase-1, lipoxygenase-2, and/or lipoxygenase-3, including any two or all three of these lipoxygenases, is absent. The compositions of the present invention may also be defined as having the following color characteristics: according to wherein L^*Represents lightness and b^*CIE-L representing the hue on the blue (-) -yellow (+) color axis^*a^*b^*The system monitors and measures b^*A value less than 30 and L^*The value is greater than 80. In still other embodiments, the compositions provided herein can comprise 67-69mg lysine per gram of protein, can comprise 72-80mg arginine per gram of protein, and/or can comprise 28-30mg histidine per gram of protein.

In yet another aspect, the present invention provides a method of analyzing the odor-generating characteristics of soybeans, the method comprising determining the level of at least one compound selected from the group consisting of 2, 4-decadienal, hexanol, hexanal, and 1-octen-3-ol. In one embodiment, the method can include determining the level of the compound, the determining including incubating a mixture of about 1 part soybean seed meal and about 4 parts water for a time period of about 1 to about 40 minutes, and quantifying the amount of at least one compound selected from the group consisting of 2, 4-decadienal, hexanol, hexanal, and 1-octen-3-ol, and combinations thereof, with a deuterated standard of hexanal, hexanol, and decadienal. The soybean seed powder may be obtained from dehulled soybeans.

In yet another aspect, the present invention provides a method of obtaining a soybean variety that produces soybeans and soybean pulp with reduced odor-producing characteristics, the method comprising measuring the level of at least one compound selected from the group consisting of 2, 4-decadienal, hexanol, hexanal, and 1-octen-3-ol, and any combination thereof, in one or more soybeans or soybean pulp from first and second soybean varieties, and selecting a variety that produces seeds with lower levels of the compound. The method can further comprise crossing a plant of the selected variety with another plant to produce a progeny, and measuring the level of at least one compound selected from the group consisting of 2, 4-decadienal, hexanol, hexanal, and 1-octen-3-ol, and any combination thereof, in one or more soybeans or soybean pulp from the progeny.

In yet another aspect, the present invention provides a method of selecting a soybean variety that is resistant to fungal infection, the method comprising selecting a variety comprising less than 5 μ g 1-octen-3-ol per gram of seed as determined by incubating a mixture of about 1 part soybean seed meal and about 4 parts water for a time period of about 1 to about 40 minutes and measuring 1-octen-3-ol.

In yet another aspect, the present invention provides seed of soybean plant of designation 0119149, representative seed of said plant having been deposited with the ATCC under accession number PTA-6197. The invention further provides a soybean plant 0119149 or parts thereof produced by growing such seeds. Such plants of the invention may comprise a transgene. In still other embodiments, the present invention provides a method of producing a soybean plant derived from the soybean plant 0119149, the method comprising the steps of: (a) preparing a progeny plant derived from soybean plant 0119149 by crossing a plant of soybean plant 0119149 with another soybean plant, wherein a seed sample of soybean plant 0119149 has been deposited with the ATCC under accession No. PTA-6197; (b) crossing the progeny plant to itself or to a second plant to produce seed for a next generation progeny plant; (c) growing a next generation progeny plant from the seed and crossing the next generation progeny plant to itself or to a second plant; and (d) repeating steps (b) and (c) for additional 3-10 generations to produce an inbred soybean plant derived from soybean plant 0119149.

In yet another aspect, the present invention provides soybeans having improved organoleptic properties (i.e., soybeans with improved taste, color, odor, and mouthfeel characteristics) after oxidation under mild aqueous conditions. Also provided are lighter colored soybeans to improve the organoleptic properties of the soybeans. Soybeans with low levels of free arginine and asparagine are further provided to improve the organoleptic properties of soybeans. In another embodiment, soybeans with reduced levels of linoleic and linolenic acids are provided to improve organoleptic properties.

In one embodiment, the soybean plants provided herein can comprise one or more transgenes. Examples of transgenes include genes that confer herbicide resistance (which results in plants with herbicide resistance) and genes that confer insect resistance.

According to the present invention there is provided soybean seed comprising lipoxygenases 1, 2 and 3, comprising more than about 10% linoleic acid as a percentage of total fatty acids, which upon oxidation under mild aqueous conditions yields less than 20 μ g of total 2, 4-decadienal plus hexanal plus hexanol per gram of ground seed.

According to another aspect of the present invention there is provided soybean comprising lipoxygenase with less than about 4% linolenic acid and greater than about 10% linoleic acid as a percentage of total fatty acids that, upon oxidation under mild aqueous conditions, yields less than 20 μ g of total 2, 4-decadienal plus hexanal plus hexanol per gram of ground seed. The same soybeans, when oxidized under mild aqueous conditions, also produce 1-octen-3-ol contents of less than 8 μ g/g of ground seed.

In accordance with yet another aspect of the present invention, there is also provided soybeans comprising less than about 2000 μ g of free arginine and less than about 400 μ g of free asparagine per gram of dry seed weight that, upon oxidation under mild aqueous conditions, yield less than about 20 μ g/gm of 2, 4-decadienal, hexanal, and hexanol per gram of ground soybeans. The same seeds, after oxidation under mild aqueous conditions, may also produce a 1-octen-3-ol content of less than 8 μ g per gram of ground soybean seeds.

According to yet another aspect of the present invention there is provided soy beans having a yellow color of "b^*The value measures less than 30, yielding less than 20 μ g/gm of 2, 4-decadienal, hexanal, and hexanol per gram of ground soybeans after oxidation under mild aqueous conditions. The same seeds, after oxidation under mild aqueous conditions, may also produce a 1-octen-3-ol content of less than 8 μ g per gram of ground seeds.

According to yet another aspect of the present invention, there is provided soybean having greater than 30% of the protein as β -conglycinin and less than 25% of the protein as glycinin that produces less than 20 μ g/gm of 2, 4-decadienal, hexanal and hexanol per gram of ground soybean after oxidation under mild aqueous conditions. The same seeds, after oxidation under mild aqueous conditions, may also produce less than 8 μ g of 1-octen-3-ol per gram of ground seeds.

In accordance with the present invention, there is provided soybeans comprising less than 5,000 μ g of free arginine per gram, less than 900 μ g of free asparagine, greater than 30% of the protein is beta-conglycinin, and less than 25% of the protein is glycinin that produce less than 20 μ g/gm of 2, 4-decadienal, hexanal, and hexanol per gram of ground soybeans after oxidation under mild aqueous conditions. The same seeds, after oxidation under mild aqueous conditions, may also produce less than 8 μ g of 1-octen-3-ol per gram of ground seeds.

According to yet another aspect of the present invention, there is provided soybean produced by crossing a first soybean seed having greater than 30% total protein as β -conglycinin and less than 25% protein as glycinin with a second soybean seed that, after oxidation under mild aqueous conditions, produces less than 20 μ g/gm of 2, 4-decadienal, hexanal and hexanol per gram of ground soybean.

According to yet another aspect of the present invention there is provided soybean produced by crossing a first soybean seed comprising less than 4% linolenic acid and greater than 10% linoleic acid as a percentage of total fatty acids with a second soybean seed comprising lipoxygenases 1, 2 and 3 comprising greater than 10% linoleic acid as a percentage of total fatty acids, which upon oxidation under mild aqueous conditions produces less than 20 μ g/gm of 2, 4-decadienal, hexanal and hexanol per gram of ground soybean.

According to yet another aspect of the present invention, there is provided a method of analyzing odor-generating characteristics of soybean seed varieties, the method comprising incubating a mixture of about 1 part soybean seed meal and about 4 parts water for a time period of about 1 to about 40 minutes, and quantifying the amount of at least one compound selected from the group consisting of 2, 4-decadienal, hexanol, hexanal, and 1-octen-3-ol, and combinations of two, three, or four thereof, with a deuterated standard of hexanal, hexanol, and decadienal.

According to yet another aspect of the present invention, there is provided a soybean cultivation method comprising incubating a mixture of about 1 part soybean meal or dehulled soybean meal and about 4 parts water at room temperature for a time of about 1 to about 40 minutes, and quantifying the amount of 2, 4-decadienal, hexanol, hexanal and 1-octen-3-ol with deuterated standards of hexanal, hexanol and decadienal, and selecting seeds from a breeding population according to the results.

According to another aspect of the invention, there is provided soybean comprising a transgene, such as an herbicide resistance gene that confers herbicide resistance and an insecticidal gene that confers insect resistance.

According to another aspect of the present invention there is provided a processed food for human consumption comprising soy bean having greater than 30% of the protein as beta-conglycinin and less than 25% of the protein as glycinin, which upon oxidation under mild aqueous conditions yields less than 20 μ g/gm of 2, 4-decadienal, hexanal and hexanol per gram of ground soy bean.

Description of the exemplary embodiments

The present invention provides soy compositions, soy beans and soy bean seed derivatives having improved organoleptic properties, and methods for their production. The soy compositions of the present invention provide improved taste, color, odor and mouthfeel characteristics. The invention also provides methods of producing such compositions, and methods of determining the ability of a soybean variety to produce the major odors identified as 2, 4-decadienal, hexanal, hexanol, and 1-octen-3-ol and using the results to select seeds from a breeding population.

According to the invention, the oxidation conditions can be generated as follows: about 0.5 parts soy flour is mixed with 2mL of water or 1 part soy flour is mixed with 4 parts water to disperse the solid particles in water and allow the oxidation reaction to occur for about 1-40 minutes at room temperature, which may be between 15 ° and 40 ℃. The concentrated suspension allows enzymes, substrates, free radicals, free radical scavenging compounds, enzyme inhibitors and other factors to influence the amount of odor generated.

The present invention provides lipoxygenase-containing soybeans comprising less than 4% linolenic acid and/or greater than 10% linoleic acid as a percentage of total fatty acids that, upon oxidation under mild aqueous conditions, produce less than 20 μ g of 2, 4-decadienal (CH) per gram of ground seed, and compositions derived therefrom₃(CH₂)₄CHCHCHCHCHCHO, CAS No.25152-84-5) plus hexanal (CH)₃(CH₂)₄CHO, CAS No.66-25-1) plus hexanol (CH)₃(CH₂)₅OH, CAS No. 111-27-3). The same soybeans, when oxidized under mild aqueous conditions, also produce less than 8 μ g of 1-octen-3-ol (CH) per gram of ground seeds or dehulled soybean meal₃(CH₂)₄CHOHCHCH₂CAS No. 3391-86-4). The compounds 2, 4-decadienal, hexanol, hexanal and 1-octen-3-ol and combinations thereof were used to quantify the odor generating properties of soybeans. The odor is not limited to the compounds listed. Other detectable aldehydes, ketones and alcohols can also be used as a measure of odor-generating properties using the methods of the present invention. Examples of such compounds include, but are not limited to, propionaldehyde, pentenal, pentanal, hexenal, pentenol, heptanal, heptenal, benzaldehyde, adienal, heptadienal, heptanol, octenol, octenal, nonanal, octadienone, 2-pentylfuran, 2, 3-dimethylpentanal, nonenal, maltol, decenal, and 2-undecenal. According to the present invention, the term "lipoxygenase" refers to an enzyme that catalyzes the oxidation of unsaturated fatty acids with oxygen to produce peroxide. The term "lipoxygenase" (ec.1.13.11.12) is also known in the art as lipoxygenase and dioxygenase. The odor of soymilk and soy protein components from soybeans lacking one, two or three of the lipoxygenases 1, 2 and 3 was evaluated by other researchers. High oleic soybeans present less than 4% linoleic acid. It was found with the assays of the present invention that certain soybeans having these traits could produce less than 20 μ g per gram of ground soybeansTotal 2, 4-decadienal plus hexanal plus hexanol, while some do not fall within this range. It has been found in the present invention that it is possible to construct soybeans containing lipoxygenases 1, 2 and 3 with very low levels of odor generation, and that lipoxygenase-free soybeans may also produce high levels of off-flavors. In addition to high β -conglycinin compositions that have not previously been screened for odor-generating properties, the present invention specifically provides novel soy compositions containing lipoxygenases 1, 2 and 3 and more than 10% linoleic acid. According to the invention, linoleic acid (18: 2n-6) and linolenic acid (18: 3n-6) are polyunsaturated fatty acids with two or three cis double bonds. The method of selecting low odor producing lines from progeny of lipoxygenase-free soybeans or high oleic soybeans of the present invention falls within the scope of the present invention.

The odorous 1-octen-3-ol is a split product of fatty acids with hydroperoxide at the 10 carbon position of linoleic acid. The present inventors have discovered that removing the hulls greatly reduces the 1-octen-3-ol forming characteristics of the soy composition. Fungal lipoxygenases and hydroperoxide lyases form 10-hydroperoxide and 1-octen-3-ol, respectively (Wurzenberger and Grosch, 1984; Husson et al, 1998). It is theorized by the present invention that soybeans with lower 1-octen-3-ol production are resistant to soybean hull contamination by fungi such as fungi of the genus Phomopsis (Minor et al, 1995) and/or contain ingredients that inhibit fungal lipoxygenase.

The organoleptic properties of soy products depend on the content of glycinin and beta-conglycinin. Glycinins tend to retain odor and tend to form insoluble particles that adversely affect the organoleptic qualities of the soy product. The present invention provides soybeans having greater than 30% of the protein as beta-conglycinin and less than 25% of the protein as glycinin that produce less than 20 μ g of total 2, 4-decadienal plus hexanal plus hexanol per gram of ground seeds after oxidation under mild aqueous conditions. The same seeds may also produce less than 8 μ g of 1-octen-3-ol per gram of ground seed under similar conditions. According to the invention, beta-conglycinin refers to a protein trimer having a molecular weight of 150-200 kDa. The three major subunits of β -conglycinin are α' (72kDa), α (68kDa) and β (52 kDa). The α' and α subunits contain two covalently bound carbohydrate moieties, and the β subunit contains one. An overview of the structure and properties of beta-conglycinin and another major storage protein, glycinin, is given by Utsumi et al, (1997). The ratio of alpha' to alpha subunit of beta-conglycinin can be altered using traditional breeding methods using a common germplasm such as "Moshidou Gong 503". The term β -conglycinin is used in this application to include these subunit variants. Seeds of the invention having greater than 30% of the protein as beta-conglycinin and/or less than 25% of the protein as glycinin are provided, which comprise less than 5,000 μ g/g free arginine and less than 900 μ g/g free asparagine. The term "free" refers to an amino acid that does not bind to other molecules present in the soybean or soybean seed meal, which can be dissolved by extraction with 5% trichloroacetic acid (TCA) in water at 4 ℃ overnight. The value of selecting soybeans that contain low levels of free amino acids to produce high quality soybean pulp, soymilk, soy flour, soy protein concentrate, and soy protein isolate has not been previously demonstrated.

The color of the soy ingredients and food products made according to the present invention can be improved by reducing the levels of aldehydes formed (e.g., hexanal and 2, 4-decadienal) as the aldehydes react with amines to form brown pigments. The reduced level of lipid oxidation products also limits the oxidative bleaching of the yellow pigment, making the final product less white in color. This potential low bleaching problem is addressed in the present invention by selecting soybeans that contain low levels of yellow pigment. The present invention provides a soy composition having a yellow color according to "b^*The value "measures less than 30, yielding less than 20 μ g of total 2, 4-decadienal plus hexanal plus hexanol per gram of ground seeds after oxidation under mild aqueous conditions. "b" as used herein to describe the color of soybean seeds^*"value represents CIE-L^*a^*b^*The color scale (CIE, Colorimetry, Publication 15.2, Second Edition, Vienna (1986), using Colorflex procedure) refers to the blue (negative) to yellow (positive) color of soybean or soybean meal, and similarly, "L^*"value meansIn CIE-L of soybean or soybean powder^*a^*b^*Lightness on the color scale. In one embodiment of the invention "L" of soybean or soybean meal^*"value greater than 80.

Seeds comprising the low odor producing traits plus other desirable traits (e.g., yield, high β -conglycinin composition, herbicide resistance) can be constructed by crossing the desired wild type soybean, commercial cultivar or hybrids thereof with soybean plants whose seeds have the low odor producing phenotype of the invention by conventional plant breeding methods. Progeny of the cross that exhibit the low-odor trait and other desired phenotypes are then selected. Breeding methods used according to the invention include, for example, the methods described by Knowles and Briggs (1967) and any similar method known in the art. Specific methods for selecting and developing new soybean varieties are disclosed, for example, in U.S. patent No.6,653,534.

The present invention also provides processed foods for human consumption prepared from the soybean composition of the present invention. Examples of processed foods for human consumption may be prepared, for example, from the dehulled soy flour compositions of the present invention. Examples of such derivatives include, but are not limited to, bars (bars), beverages, meat and meat substitutes, soy yogurt, cheese substitutes, nutritional supplements.

The following examples are included to illustrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain substances which are both chemically and physiologically related may be substituted for the substances described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Example 1

Materials and methods

This example describes the analytical method of the present invention. The purpose of this analytical method was to determine the odor-generating properties of different soybean lines.

The method measures a selected odor by first initiating odor formation. Grinding the seeds into fine powder and activating enzymes with water to promote the formation of odor. Studies of the rate of formation showed that the formation of odor at room temperature was substantially complete after about 20 minutes (table 1). This time was important for successful quantification of the odorant compounds and evaluation of the odor-generating properties of the different soybean lines. Deuterated substitutions of hexanal, hexanol, and 2, 4-decadienal were added after 20 minutes to provide internal standards. Sodium sulfate was added to stop the reaction, followed by 10% methanol to ether to extract aldehydes, alcohols and ketones. The method is not limited to the compounds listed. All other detectable aldehydes, ketones and alcohols can be quantified, but not as precisely as the three above-mentioned odors using deuterated substitutes.

Table 1: effect of the time after mixing of Soybean flour with Water (0.5g flour 2mL water, i.e., 1: 4 ratio) on flavor development. The pH of the suspensions of the four different soybean lines (A-4, A-5, A-10 and A-14) was about 6.3.

Time (minutes)	Hexanal (mu g/g)	2x stdev.	1-Octen-3-ol (μ g/g)	2x stdev.	2, 4-decadienal (ug ^ g)	2xstdev.	pH(A-4)	pH(A-5)	pH(A-10)	pH(A-14)
											05102040240	1.311.811.414.015.717.0	2.60.21.40.81.94.2	0.82.93.75.87.88.9	0.4040.70.60.51.8	0.46.96.57.88.69.9	0.40.70.60.92.31.7	6.46.36.36.3	6.46.36.36.3	6.36.36.36.3	6.36.36.36.3

The analysis time to determine these components in 175 samples took 24 hours. This includes extraction and measurement by gas chromatography/mass spectrometry. The size of the sample for analysis is usually 0.5 grams (g), but may be in the range of 0.2g to 0.7 g. The odor concentration ranges from 0.2. mu.g/g to 120. mu.g/g on a wet weight basis.

Preparation of soybean pulp sample: whole seeds or soybean pulp were collected as samples. Approximately 6-12 randomly selected seeds or comparable weight pieces of soybean pulp from the samples were ground in a ball mill at approximately 1200 revolutions per minute (rpm) for 1 minute to produce a fine powder. Ball mills for grinding seeds are described in U.S. patent publication 2003/0146313a 1. The amount of seeds or soybean pulp is determined to obtain about 0.5-1.0g of soybean meal at the end of milling. Freshly ground soy flour was used for further analysis.

Extraction of soybean meal: freshly ground soy flour was used to extract the main odor. About 0.5g (0.48-0.52g) of soy flour was placed in a20 milliliter (ml) vial with a cap (VWRTRACECLEAN)^TMA clear borosilicate glass teflon-lined sealed bottle). Deionized water was added to the soy flour in the vial and the cap was then closed back. The contents of the vial were mixed in a vortex mixer for about 30 seconds to ensure that all the soy flour was fully hydrated in the vial. The hydrated soy flour is incubated under mild aqueous conditions, defined herein as 20 minutes incubation in water at room temperature (22 ℃). After 20 minutes, 11 ± 0.3 grams of anhydrous sodium sulfate was added to the vial, followed by 10ml of a 10% methanolether solution. Then 30 microliters of standard surrogate (mixture of deuterated standards of hexanal, hexanol, and decadienal) tracer solution (spiking solution) was added to the vial, followed by capping and shaking on a reciprocal shaker at 200rpm for 30-40 minutes. After 30-40 minutes, 1ml of the methanolether extract was placed in an autosampler sample vial (7683 autosampler sample vial for HP autosampler, VWR as vendor) for further analysis.

Analysis of soybean meal extract: extracting soybean flour with methanol/ether extraction medium in Agilent 6890 gas phase equipped with Agilent 7683 series automatic samplerFurther analysis was performed in a chromatograph (395Agilent Technologies, Palo Alto CA 94306) and a Leco Time of Flight mass spectrometer with LECO Chrom TOF software (LECO Corporation, St. Joseph, Michigan 49085). The gas chromatograph was also equipped with a 10 meter long DB-WAX or DB1701 gas chromatography column, with a film thickness of 0.4 or more and an internal diameter of 0.18mm (Agilent technologies). Methanol (methanol is EM Science purge trap grade methanol) was obtained from VWR (VWR International, West Chester, PA 19380); ether (dry ether) was obtained from Mallinckrodt (Mallinckrodt, Hazelwood, MO 63042). 2, 4-decadienal, 85% trans (15% cis), hexanal 98%, hexanol 99%, 1-octen-3-ol 98%, 2-undecanone 99%, 2-nonenal 97% and 2, 4-nonenal 99% were obtained from Sigma-Aldrich company (Saint Louis MO 63103). Deuteration D₁₂Hexanal, D₁₃Hexanol and D₂2, 4-decadienal was prepared in-company as described by Lin et al (1999). To analyze the sample, 1 microliter (μ l) of the sample was injected through the injection port of the gas chromatograph (gc). The parameters used in the gas chromatography to analyze the samples were as follows: a chromatographic column: DB-Wax or DB1701 capillary column 10m x 0.18mm, 0.4mm membrane, injection volume: 1ul, injection cannula (liner): split/no-split sleeves.

Temperature program: initial 55 ℃ for 1 minute, 40 ℃ to 175 ℃, 40 ℃/min, hold for 0 minute, 175 ℃ to 240 ℃,35 ℃/min, hold for 0 minute, inlet temperature: 220 ℃, injection mode: pulse no split, initial 8psi 1.5 min split ratio: 20: 1, carrier gas: helium, constant flow of 1.8 mL/min.

LECO time-of-flight parameter temperature interface: 250 ℃; source temperature: 200 ℃; source temperature of mass spectrometer: 150 ℃; scanning parameters are as follows: 50-250m/z, about 50 scans per second.

Quality control of the analysis: for each batch of samples, the method blank and addition analysis (spike) were run simultaneously. The additive analysis is performed by dividing one of the samples to be analyzed into two parts. The sample should be as homogeneous as possible. The second part adds known amounts of hexanal, 1-octen-3-ol, and decadienal. This addition is made at the time of addition of the deuterated compound in the extraction procedure described above. The concentrations of added and non-added compounds were determined as% recovery according to the following formula:

wherein:

C_sthe concentration of the added sample, in μ g/(g wet weight)

C₀The concentration of the sample added is in units of μ g/(g wet weight)

Wt_sConcentration of added sample in grams

X_sMicrogram added to sample

The process blank was performed according to the extraction procedure, but without the addition of soy flour.

Accuracy and precision of the analysis: accuracy and precision were determined by running a uniform sample of soy flour and adding known levels of the three compounds hexanal, 1-octen-3-ol, and 2, 4-decadienal to the sample. The non-added samples were also analyzed to determine the amount of added compound recovered. Different levels of addition can be used as standard addition methods to determine the background caused by systematic errors. The mean and standard deviation were determined. The average value is compared to known levels of added material to assess the accuracy of the method. The standard deviation gives the precision of the measurement. It is important to note that these methods only measure the differences analyzed, since grinding more than 40 seeds to produce a uniform soy flour averages the seed differences. Larger differences may occur for actual measurements due to seed-to-seed differences. The% recovery for hexanal was 83.8%. The% recovery for 1-octen-3-ol was 93.6%. The% recovery for 2, 4-decadienal was 99.3%. Hexanal is least accurate by addition. This is believed to be due to the volatility of hexanal and the fact that the process has a low recovery of hexanal. The accuracy of the addition method of the octen-3-ol and the 2, 4-decadienal is higher. Precision expressed as% relative standard deviation of recovery values was 5% for hexanal and octen-3-ol. For 2, 4-decadienal, the precision as measured by the% relative standard deviation of the recovery values was 1.1%. For this homogeneous sample, no effect was found for sample sizes from 0.2 to 0.9 grams. Sample size can be a problem between samples, however, this has not been tested. The sample size should therefore be about 0.5 grams before it is known that sample size is not an issue or adjustments are made to the process.

For all compounds, the detection range was 0.5 to 25 micrograms. Adding more standards to the standard curve can expand this range.

Example 2

Identification and selection of low odor producing soybeans

The strong odor of soymilk made from control soybeans (Vinton 81), soybeans lacking lipoxygenase-2 (QT-1) and soybeans lacking lipoxygenases 1, 2 and 3 (IA2025) was quantified. Soymilk was prepared from each soybean variety by soaking the desired clean seeds in water at 25 ℃ for about 8 hours at a ratio of 1: 5 (1g weight: 5ml water). After discarding the soaking water, twice the original weight of soybeans was drained and mixed with freshly distilled water (2X dry weight of soybeans) for five minutes. Additional distilled water (7X dry weight of soybeans) was mixed into the resulting slurry at 20 ℃ for about 2 minutes. The slurry was simmered in a water bath at 95-98 ℃ for 20 minutes, then filtered through a coarse woven cloth wrapper (wovencheeseciloth) and squeezed by hand to squeeze out as much soymilk as possible. The soymilk was pasteurized by simmering in a water bath at 85-90 deg.C for 10 minutes to reduce microbial contamination, and then stored at 4 deg.C for further analysis of extracted odors.

Extracting odor from the soymilk sample. With 0.67 part Freon^TM113 extracting the soymilk for at least 30 minutes. Removal Freon^TM113 after extraction, the aqueous phase is further extracted with 0.67 parts of ethyl acetate. CollectAfter extraction with ethyl acetate, the aqueous phase was discarded. Freon will be^TMAnd ethyl acetate was filtered over magnesium sulfate to remove as much water as possible, then concentrated to 1ml with a Buchi 0.1 rotary evaporator. Freon extract was evaporated at 48 kilopascals (kPa) and ethyl acetate extract was evaporated at 86 kPa.

The strong odor produced in soymilk was quantified by GC-odor assay (Acree T.E, analytical chem.69: 170A-175A, 1997). GC-odometry is a gas chromatograph with an olfactory port (sniffing port) that can measure the efficacy (potency) of a chemical compound as an odorant (odorant) as a measure of a human reaction to the odorant in the air stream or breath. A Hewlett Packard 6890 gas chromatograph equipped with a 12m × 0.32mm cross-linked polymethylsiloxane fused silica capillary column (film thickness 0.33 μm) was used for Charminalysis^TM(Acree, T.E.; Bamard, J; Cunningham D.G.; FoodChem.14, 273-. The chromatography effluent consisted of helium (2ml/min) as a carrier gas and nitrogen (about 30ml/min) as a make-up gas. The chromatography effluent was mixed with inhaled air (sniffing air) (20L/min, 99% laboratory air, humidity adjusted to between 50% and 75%) and passed through an olfactory analyzer via a silylated pyrax tube of 10mm diameter. The GCMS furnace program was set to start increasing its temperature from a starting temperature of 35 ℃ to 225 ℃ at a rate of 6 ℃/min. To charman analysis^TMFor more details on the method used for soymilk, see doctor's academic paper submitted by Yu-Wen Feng to the university of cornell institute of graduates.

Both soybeans lacking lipoxygenase-2 (IA2025, QT-1) produced lower levels of hexanal than the control soybeans (Table 2). Soybean lacking all three lipoxygenases (IA2025) produced the highest levels of 2, 4-decadienal and 1-octen-3-one, whereas soybean lacking lipoxygenase-2 (QT-1 variety) had the lowest levels of all five strong odors (Table 2). As can be seen from the results, there are unknown compositional factors other than lipoxygenase-2 involved in controlling lipid oxidation in soybeans. The QT-1 soybean variety was identified as a useful variety for constructing commercial low-odor soybean varieties that may or may not contain lipoxygenase-2. The method of example 1 was developed to identify progeny of QT-1 and other soybean lines that produce low levels of 2, 4-decadienal, hexanal, hexanol, and 1-octen-3-ol.

Table 2: the odor attractiveness value (charrvalue) present in soymilk prepared from three varieties of soybeans. The Monsanto line produces lower levels of off-flavors than soybeans without the three lipoxygenases (IA2025) and tofu soybeans (Vinton 81).

Soybean line	1-octen-3-one	2, 4-decadienal	E, Z-2, 6-nonadienal	E-2-nonenal	Hexanal
						Low odour strain QT-1	3	25	11	67	69
IA2025Vinton 81	18265	20871	168186	165165	73224

Table 3 describes soybean hybrids, which are used to produce progeny that describe the present invention. These lines were generated using standard plant breeding methods.

Table 3: a soybean hybrid is produced that is useful for the generation of progeny that describe the soybeans and methods of the invention.

Hybrid body	Type of hybridization
		MonQT-1/A346E＞A2247/6P24S	AB
A2533/IA2027	C
		IA2032/A3469CP3469/1A2025	DE

Example 3

Low odor producing, Low color and Low free amino acids in Soybean lines selected according to the invention Demonstration of annual and site-to-site correspondence of traits

For color evaluation, whole seeds were collected as samples. The desired number of seeds was selected from the sample and ground in Mega-Grinder at 1200rpm for 1 minute to produce ground soy flour. Mega-Grinder used to grind seeds is described in U.S. patent publication 2003/0146313A 1. Freshly ground soy flour was used for further analysis.

The color of the soy flour was measured using a ColorFlex Spectrocolorimeter Model 45/0 color measurement system manufactured by Hunter labs (Hunter Associates Laboratory Inc, Reston VA, USA) following standard procedures recommended by the manufacturer. Using the ColorFlex program, according to CIE-L^*a^*b^*The color scale (CIE, Colorimetry, publication 15.2, second edition, Vienna, 1986) measures color. The international Commission on illumination (international Commission on illumination, abbreviated CIE under its french name Commission on international' Eclairage) is an organization devoted to the international collaboration and communication of information between its member countries about all matters relating to lighting science and technology. L is^*The value refers to the lightness of the soy flour, and b^*Values refer to the blue (negative) to yellow (positive) color of the soy flour. Color values of soy flour made from different lines are shown in tables 4 and 5.

Free amino acids: the unground samples were kept in a temperature/humidity controlled closed (APHIS approved) room. The samples were ground with CAT Mega-Grinder to produce soybean meal, which was stored in a seed storage room at 4 ℃. Extracting soybean flour with 5% TCA overnight at 4 deg.C, and storing the extract at-80 deg.C. The extract was filtered, diluted if necessary, and then analyzed for free amino acids by the OPA method. OPA method the samples were derivatized using ortho-phthalaldehyde (OPA) prior to injection onto a C18 reverse phase HPLC column. The derivatized base amino acid can be efficiently separated by the R group and quantitatively detected by a sensitive fluorometer. The relative standard deviation of this method is-3%.

Lipoxygenase activity: the samples were ground with Mega-Grinder. Each freshly ground sample was weighed out in triplicate (5 mg. + -. 1) and extracted in 2-ml 96 wellsIn a specific hole of the plate. With 0.1MK₂HPO₄(pH7.0 or pH9.0) the sample was extracted at room temperature for 1 hour. The resulting supernatant after centrifugation was used to measure the consumption of linoleic acid (substrate) spectrophotometrically, and then the total protein of each sample was determined using the Bio Rad protein dye. The change in absorbance at 230nm during the reaction was measured for 1 minute and the extinction coefficient (ε 23,000M)^-1cm^-1) The lipoxygenase units were calculated. The substrate concentration consumed during the reaction was calculated by substituting each value into equation a ═ epsilon bC. One unit of lipoxygenase is defined as the number of μmoles of substrate consumed per minute per mg of total extracted protein. This assay can measure the level of lipoxygenase-2/-3 or lipoxygenase-1 (enzyme units), respectively, using reagent solutions prepared at pH7.0 or pH 9.0. The results for lipoxygenase units are given in LOX units pH7.0 for lipoxygenase-2 and 3 activities and in LOX units pH9.0 for lipoxygenase-1 activities.

As a result: although soybeans were grown in multiple locations (table 4) and years (tables 4 and 5), the soybean odor profile persists. The soybean lines that produced low levels of hexanal, hexanol and 2, 4-decadienal, and 1-octen-3-ol in the odor assay (described in example 1) were consistently different from the lines that produced high levels (table 4). The soybean lines in table 4 were ranked in order of their hexanal + hexanol +2, 4-decadienal levels produced. For example, line A-1 at the top of the table produced 18.21 +/-4.21. mu.g/g of the three odors, in contrast to line A-18 at the bottom of the table, which produced 65.74 +/-21.97. mu.g/g of the three odors. The amount of hexanal plus hexanol produced by soybeans from the same hybrid (e.g., hybrid type A) correlated with the amount of 2, 4-decadienal produced (Table 6, R)²0.85), suggesting similar mechanisms and controls for genetic and compositional changes. The 1-octen-3-ol level produced was independent of hexanal, hexanol and 2, 4-decadienal (Table 6, R)²0.01), which suggests different mechanisms of formation and control. Soybean lines producing low levels of 1-octen-3-ol and lines producing high levels of 1-octen-3-ol cultivated in 2-3 sites are consistently different. For example, line A-18 produced 4.70 +/-0.88. mu.g/g of 1-octen-3-ol, while line A-12 produced 14.67+/-2.37. mu.g/g of 1-octen-3-ol. Lines can be selected that have a combination of genetic compositions that produce lower levels of 1-octen-3-ol and lower levels of 2, 4-decadienal plus hexanal plus hexanol (e.g., a-6).

Tables 4A and B: color and odor resulting from ground soybeans made from progeny of hybrid types A, B, C, D and E shown in Table 3. Each value is the mean and standard deviation (Stdev) of each line grown in 2002 at two or three sites (Ames, Iowa; Oxford, Indiana; Gladbrook, Iowa). The progeny of the listed hybrid type B had a low linolenic acid content of 2.9 +/0.4% of the total fatty acids. All lines were scraped of yellow hilum. The moisture content of the ground soybeans was 8%. Color value of L^*(brightness of light), a^*(Green-Red) and b^*(blue-yellow). Abbreviations: stdev ═ standard deviation.

Table 4 a.1:
													type of hybridization	Hexanal (mu g/g)	Hexanol (μ g/g)	Hexanal + HexOH (μ g/g)	1-Octen-3-ol (μ g/g)	2, 4-decadienal (μ g/g)	2, 4-decadienal + hexanal + hexanol (μ g/g)	Hexanal (. mu.g/g) (Stdev)	Hexanol (. mu.g/g) (Stdev)	Hexanal + Hexanol (. mu.g/g) (Stdev)	1-Octen-3-ol (. mu.g/g) (Stdev)	2, 4-decadienal (. mu.g/g) (Stdev)	2, 4-decadienal + hexanal + hexanol (Stdev)
C1, L-absent	9.78	1.06	10.84	10.79	6.23	17.07	3.36	0.93	4.29	0.34	0.64	4.93
													A-1	9.80	1.89	11.68	9.14	6.53	18.21	1.35	1.04	2.38	0.14	2.16	4.54
B-1	9.88	0.34	10.22	10.03	8.52	18.75	3.93	0.48	4.42	3.39	0.06	4.48
													D1, L-absent	12.59	0.92	13.51	13.51	5.24	18.75	1.89	0.83	2.16	3.11	2.46	4.58
A-2	11.86	1.25	13.11	8.59	6.55	19.66	2.15	1.09	3.23	2.90	0.63	2.64
													B-2	11.99	1.78	13.76	8.19	6.91	20.67	4.07	1.11	5.18	2.98	1.37	6.55
A-3	12.60	1.53	14.12	10.87	7.29	21.41	3.23	0.23	3.45	3.48	2.09	5.34
													A-4	14.76	2.30	17.05	9.29	6.32	23.38	2.71	0.23	2.94	2.16	1.29	4.01
A-5	14.95	1.38	16.33	9.08	7.65	23.98	4.94	0.17	5.08	1.02	4.55	9.55
													A-6	17.5S	3.21	20.79	4.63	8.97	29.75	1.26	1.36	2.62	2.90	5.38	2.76
A-7	19.3S	3.35	22.73	7.71	8.39	31.12	3.03	1.76	3.49	1.54	2.69	3.73
													A-8	23.69	2.73	26.42	9.14	11.51	37.93	10.64	0.69	10.90	3.53	4.64	15.49
A-9	25.45	3.41	28.86	10.12	10.53	39.39	6.71	0.52	6.25	0.93	1.70	7.60
													A-10	27.14	3.11	30.25	10.24	9.92	40.17	4.99	2.04	6.19	1.31	2.73	7.86

Table 4 a.2:
													type of hybridization	Hexanal (mu g/g)	Hexanol (μ g/g)	Hexanal + HexOH (μ g/g)	1-Octen-3-ol (μ g/g)	2, 4-decadienal (μ g/g)	2, 4-decadienal + hexanal + hexanol (μ g/g)	Hexanal (. mu.g/g) (Stdev)	Hexanol (. mu.g/g) (Stdev)	Hexanal + Hexanol (. mu.g/g) (Stdev)	1-Octen-3-ol (. mu.g/g) (Stdev)	2, 4-decadienal (Mg' g) (Stdev)	Decadienal + hexanal + hexanol (. mu.g/g) (Stdev)
A-11A-12	31.2732.72	4.053.64	35.3236.36	8.7114.67	11.6215.18	46.9451.54	11.656.84	1.210.08	11.656.91	2.182.35	5.951.35	17.165.56
													A-13	35.16	4.30	39.46	10.03	12.33	51.78	8.20	0.99	9.20	3.74	2.74	11.94
B-3E	35.1634.26	3.664.41	38.8238.67	7.185.35	15.5716.27	54.3954.94	7.5910.47	0.450.24	8.0410.23	1.030.88	2.935.00	10.9715.22
													A-14	35.24	2.23	37.47	7.06	18.94	56.41	10.58	1.72	8.85	0.05	10.16	19.02
A-15A-16	40.0137.88	4.125.66	44.1443.53	8.258.17	14.8916.80	59.0260.33	0.8113.20	1.132.33	1.8614.11	1.913.43	2.394.61	4.2016.31
													D-3	49.52	2.26	52.3S	11.40	10.69	63.07	15.72	3.94	18.82	0.86	7.14	25.31
B-4B-5	41.2637.99	4.047.22	45.3045.21	6.909.04	18.0218.51	63.3263.72	12.2518.14	0.331.04	12.5819.17	1.270.01	7.677.55	20.2526.72
													A-17	42.44	4.72	47.16	9.32	16.99	64.15	5.64	1.02	5.38	2.59	6.57	11.78
D-2B-6	43.5440.64	10.714.97	54.2645.60	7.629.96	10.0818.82	64.3464.42	14.715.56	1.412.45	13.307.07	2.411.06	4.443.56	17.7410.62
													A-18	42.56	5.46	48.01	4.70	17.72	65.74	11.59	2.08	13.50	0.88	8.51	21.97

Table 4B 1:
														type of hybridization	Free Arg (μ g/g)	Free Asn (μ g/g)	Free ArgAsn (μ g/g)	Free Are. (Stdev)	Free Asn (Stdev)	L*	a*	b*	L*(Stdev)	a*(Stdev)	b*(Stdev)	Liposaccharidase Activity pH7	Liposaccharidase Activity pH7Stdev
C-1, L-is	845	185	1030	535	9	79.77	1.49	30.38	1.21	0.9	2.6	0.23	0.58
														A-1B-1D-1, L-none	18249681105	3319591	215510631196	124154189	39632	84.3580.2382.95	0.030.530.54	25.3528.3825.78	0.90.393.02	0.150.260.47	1.252.494.47	0.14	0.94
A-2B-2A-3	16219793157	32178420	194110583578	278865278	15532119	85.4281.784.84	0.180.220.27	22.1628.723.28	0.941.50.16	0.030.230.21	1.392.541.44
														A-4	1666	179	1845	687	104	85.73	0.01	22.21	0.68	0.04	1.26	1.69	0.31
A-5A-6	18601144	184183	20441327	216771	314	84.9582.77	0.050.09	24.1227.35	1.070.77	0.220.48	1.652.85	1.02	0.16
														A-7	2831	487	3318	496	137	85.06	-0.18	23.4	2.21	0.17	4.01
A-8A-9	16572458	221541	18782998	181264	4651	84.7583.31	0.180.36	24.5925.51	1.783.72	0.220.67	2.784.73
														A-10	2843	461	3304	612	170	83.82	0.3	24.54	3.65	0.59	4.14

Table 4B 2:
														type of hybridization	Free Arg (μ g/g)	Free Asn (μ g/g)	Free Arg + Asn (μ g/g)	Free Arg (Stdev)	Free Asn (Stdev)	L*	a*	b*	L*(Stdev)	a*(Stdev)	b*(Stdev)	Liposaccharidase Activity pH7	Liposaccharidase Activity pH7Stdev
A-11	1921	334	2255	519	103	83.59	0.32	25.29	3.97	0.67	5.72
														A-12	956	139	1095	540	10	83.42	0.11	26.67	1.71	0.22	2.79
A-13	654	76	729	460	11	83.52	-0.03	25.77	2.74	0.23	5.08
														B-3	1892	263	2154	1186	26	80.89	0.05	27.91	1.63	0.35	2.95
E	911	85	996	678	4	83.47	0.64	27.86	0.69	0.38	2.07
														A-14	1554	311	1865	732	66	84.24	-0.12	25.17	0.93	0.09	1.74
A-15	1847	292	2138	1045	217	83.68	0.28	25.01	3.41	0.63	4.15
														A-16	1860	305	2165	513	148	84.3	0.1	25.1	2.64	0.39	3.16	1.71	0.94
D-3	1280	188	1468	274	52	83.47	0.56	23.91	3.58	0.62	4.02
														B-4	992	127	1119	637	3	83.41	0.04	27.16	0.79	0.12	2.99
B-5	976	106	1081	514	8	82.75	0.11	28.39	0.44	0.21	2.13
														A-17	1618	241	1859	760	107	85.61	-0.04	21.93	1.18	0.11	2.68
D-2	1215	82	1298	758	6	82.76	0.57	28.69	0.59	0.32	0.19
														B-6	1430	157	1587	337	81	84.97	0.24	24.3	1.03	0.17	2.1
A-18	2250	452	2702	281	128	85.25	0.12	23.48	1.15	0.18	0.75

Tables 5A and B: ground soybean characteristics obtained from commercial soybean (control) and progeny of hybrid types A, B, C, D and E cultivated in 2001. The moisture content of the ground soybeans was 7%. The sequence of each line is as in table 4. Progeny of the listed cross type B have a low linolenic acid trait (2.9 +/0.4% of total fatty acids). Two progeny lack one or more lipoxygenases, others contain all three lipoxygenases (numbered LI, L2 and L3). Lipoxygenase activity is expressed in units of micromoles of substrate consumed per mg of protein. Color value of L^*(brightness of light), a^*(Green-Red) and b^*(blue-yellow). Abbreviations: stdev ═ standard deviation.

Table 5A.
													Type of hybridization	2, 4-decadienal + hexanal + hexanol (μ g/g)	2, 4 decadienal (μ g/g)	Has already gotAldehyde + hexanol (μ g/g)	1-Octen-3-ol (μ g/g)	L*	a*	b*	Liposaccharidase Activity pH7	Liposaccharidase Activity, pH9	Free arginine (mug/g)	Free asparagine (μ g/g)	Free Arg + Asn (μ g/g)
C-1, L-is	17.05	4.96	12.09	16.50	80.37	1.17	30.84	-0.092	0.297	314	39	353
													A-1	17.96	3.04	14.92	14.20	83.36	0.05	28.05	4.015	6.687	866	69	935
B-1D-1, L-nor	24.8012.37	5.163.60	19.648.77	9.904.40	81.7183.15	0.840.14	28.7126.58	3.4621.548	7.0721.479	10371124	5982	10961206
													A-2	17.44	4.00	13.44	8.70	83.42	0.17	26.40	2.860	15.800	2068	138	2206
B-2A-3A-4	27.1020.5816.90	5.384.421.60	21.7216.1615.3	9.909.305.30	81.5284.2583.98	0.21-0.19-0.18	28.5926.4326.09	3.5912.6834.400	4.5926.4124.752	55846623884	47240190	60549024074
													A-5A-6	15.7319.62	2.303.85	13.4315.77	4.908.60	82.5383.09	0.110.17	29.0028.98	4.6812.664	5.6245.949	2818687	16358	2981745
A-7	21.96	4.20	17.76	4.80	83.53	-0.33	26.13	4.515	6.257	2722	269	2992
													A-8A-9	16.8121.95	4.306.80	12.5115.15	6.705.90	82.6482.84	0.260.01	27.8628.64	3.8445.247	4.2784.090	21262594	108275	22332868
A-10	39.30	10.40	28.9	4.50	83.99	-0.33	27.35	5.520	4.901	4009	267	4276
													A-11A-12	27.4642.10	6.307.46	21.1634.64	4.3017.30	81.8883.60	0.54-0.25	29.3728.60	4.7484.010	5.6814.049	1641739	13979	1780817
A-13	34.05	8.58	25.47	16.60	82.80	-0.11	29.21	4.983	5.377	547	42	589
													B-3E	31.0633.98	7.438.33	23.6325.65	6.309.80	82.9983.83	0.180.29	28.8429.71	4.3044.444	4.1324.608	17611310	13590	18961400
A-14	41.75	9.81	31.94	11.20	82.98	-0.16	29.00	4.208	4.459	1025	86	1111

TABLE 5B hybridization types	2, 4-decadienal + hexanal + hexanol	2, 4-decadienal (μ g/g)	Hexanal + HexOH (μ g/g)	1-Octen-3-ol (μ g/g)	L*	a*	b*	Liposaccharidase Activity pH7	Liposaccharidase Activity, pH9	Free arginine (mug/g)	Free asparagine (μ g/g)	Free Arg + Asn (ug/g)
													A-15A-16	24.8631.99	5.709.70	19.1622.29	3.005.50	83.9882.58	-0.160.40	26.2229.04	4.2365.659	4.9256.237	26341493	207132	28411625
D-3	40.16	5.80	34.36	5.20	84.08	0.20	27.30	7.354	6.773	1561	152	1713
													B-4B-5	30.7038.59	5.7910.05	24.9128.54	7.9013.80	82.9482.76	0.24-0.02	29.6429.28	4.7243.485	4.6754.822	6693060	61239	7363300
A-17	35.38	8.90	26.48	6.00	84.50	0.53	25.86	5.253	4.888	993	73	1066
													D-2B-6	43.5544.02	5.8910.70	37.6633.32	8.6010.60	82.7584.43	-0.37-0.31	30.7026.47	5.4355.334	4.8485.708	17244594	114377	18384971
A-18	27.88	6.70	21.18	4.10	84.71	0.06	27.35	4.604	4.263	3378	185	3564
													Control 1 control 2 control 3	25.8028.1039.10	6.66.77.4	19.221.431.7	6.912.36	82.2079.8880.82	0.280.640.79	29.5729.1632.16	6.4863.9017.025	4.3676.0005.737	1078619674	8815661	1166774735
Control 4	23.70	7.4	16.3	5.4	81.53	0.39	30.89	3.220	3.575	1167	66	1233
													Control 5 control 6	27.5030.70	7.48.4	20.122.3	4.63.1	81.0681.64	1.180.37	31.1627.67	4.8474.845	6.2345.015	9611002	20381	11641083
Control 7	29.40	8.5	20.9	6.7	80.35	0.22	28.51	3.748	4.885	1177	168	1345
													Control 8 control 9	28.5027.90	99.6	19.518.3	10.48.4	79.4982.45	1.140.62	33.3727.88	7.1193.181	6.3885.730	3601209	5493	4141302
Control 10	25.80	9.7	16.1	9.9	80.70	0.90	31.03	5.768	5.715	970	117	1087
													Control 11 control 12	26.6034.00	10.111.6	16.522.4	8.36	80.4182.71	0.070.22	29.0230.63	5.7154.142	4.6704.585	12141674	200125	14151798
Control 13	28.30	13.6	14.7	14.8	80.79	1.27	32.83	3.181	5.730	1358	303	1661

Table 6: linear relationship defined by R-squared values between odors produced by soybean progeny harvests in 2001 and 2002. Linear regression was calculated from the data in tables 4 and 5. Abbreviations: h + H ═ hexanal + hexanol; d ═ 2, 4-decadienal; o ═ 1-octen-3-ol; DHH ═ 2, 4-decadienal + hexanal + hexanol.

Hybridization type A (2002)
					H+H	H+H1.00	D0.85	DHH0.99	O0.01
D		1.00	0.91	0.02
					DHH			1.00	0.01
O				1.00
					All hybridization types (01 and 02) H + H (01)	H+H(02)0.56	D(02)	DHH(02)	O(02)
D(01)DHH(01)		0.59	0.62
					O(01)				0.07

Using the method and soybeans of the present invention, it is possible to cultivate soybean varieties with excellent yield, low odor generation, and low color for the first time. For example, lines A-1, A-4 and A-6 produced 90, 90.5 and 104% of commercial Standard lines (commercial check).

There was no or very little correlation between the lipoxygenase activity at pH7 and 9 and the formation of 2, 4-decadienal + hexanal + hexanol from hydrated soy flour (R square < 0.35, table 7). The odor profile of soybeans producing significant lipoxygenase activity at pH7 and 9 (line a-1) was similar to that of the line lacking lipoxygenase activity (line C-1) (table 5). Commercially available soybeans (IA2032, from the 1999 harvest season) lacking lipoxygenases 1, 2 and 3 were also tested for lipoxygenase activity and flavor formation. For soy flour without three lipoxygenases, no lipoxygenase activity was found at pH7 and 9, but significant levels of hexanal (23.3. mu.g/g), hexanol (14.9. mu.g/g) and 2, 4-decadienal (5.8. mu.g/g) were formed in the odor assay of the invention. Following the experiments with lipoxygenase inhibitors, it was concluded that at least one additional lipoxygenase is active in the soy composition.

Light-coloured protein ingredients are favoured by the food industry, especially for dairy type products. Soybeans were grown in 2-3 locations to determine if low color soybeans could be selected. As a result, it was found that b can be obtained from ground soybean^*Value to select Low color soybeans (high b)^*The values indicate darker yellow and lighter green; table 4). For example, b of soybean A-2^*Value 22.16+/-1.39, b of Soybean A-6^*The value was 27.35+/-2.85 (Table 4). B of soybean line cultivated in 2001 and 2002^*There is a correlation between the values (R square 0.7; table 7).

Table 7: linear relationship of odor, free amino acids and color produced by the harvest of soybean progeny in 2001 and 2002 as determined by the R-squared value. Linear regression was calculated from the data in tables 4 and 5. For the 2002 data, the free amino acid correlation was derived under the exclusion of three outliers (A-4, B-5, and B-6).

	DHH(01)	Free Arg (01)	Free Asn (01)	Free Arg + free Asn (01)	a＊(02)	b+(02)	L＊(02)	L＊(01)
									lox active pH7(01)	0.34			0.01
lox active pH9(01)	0.00			0.10
									DHH(01)				0.01
O(01)				0.15
									a＊(01)					0.29
b＊(01)	0.00			0.28		0.65		0.48
									L±(01)							0.50
b＊(02)							0.79
									Free Arg (01)			0.81
Free Arg (02)		0.77
									Free Arg (02)			0.66
Free Arg + Asn (02)				0.75

In addition to genetic factors, environmental factors also affect the composition of soybeans, thereby affecting their odor-generating properties. The environmental impact was evident by comparing the odor-generating properties of the soybean lines cultivated in the two seasons of 2001 and 2002. For each line constructed, the range of 2, 4 decadienal + hexanal + hexanol was 12-44. mu.g/g in 2001 and 17-65. mu.g/g in 2002 (tables 4, 5). The 1-octen-3-ol production characteristics of soybean appear to be most sensitive to environmental factors, as evidenced by the lack of correlation between 1-octen-3-ol produced by the same line cultivated in 2001 and 2002 (R square 0.07, table 6). In contrast to this, the present invention is,the 2, 4 decadienal + hexanal + hexanol production levels of the soybean lines in 2001 and 2002 have a correlation (R)²0.62, table 6).

The amount of free arginine (Arg) and asparagine (Asp) in soybean progeny was determined. The amount of free arginine and asparagine is related (R)²0.81, table 7), total arginine plus asparagine in the range of 353-. The free arginine + asparagine in soybean is not related to the selected odor or color characteristics of soybean (R square < 0.3, table 7) and therefore it is necessary to measure the free amino acids to select lines with a combination of low 2, 4 decadienal, low color and low free amino acids. The good correlation between free arginine + asparagine in each line harvested in two different years (R square 0.8; table 7) demonstrates the feasibility of selecting low free amino acid soybean lines.

Example 4

Low odor generating properties and high beta-conglycinin composition and low free arginine And asparagine composition and low color characteristics

The source of the high β -conglycinin trait is mutant soybeans lacking glycinin and having β -conglycinin in about 55% of the total protein (U.S. Pat. No.6,171,640). As described above, the lipoxygenase assay is not useful for selecting low odor producing strains. Soybeans were constructed whose protein, fat and amino acid properties (profile) were within the normal ranges for commercial soybeans.

Quantification of soy protein subunits: about 8 seeds were ground with Mega Grinder (U.S. patent publication No. 2003/0146313A 1). For each sample, 30mg of the meal was extracted on an nutator (nutator) or multi-plate vortex shaker with 0.1M DTT in 1.0mL Laemmlis SDS buffer pH6.8 for 45-60 minutes. The tube was centrifuged for 3-5 minutes. A portion of the supernatant was transferred to a microcentrifuge tube and diluted with the above buffer to yield 1.2-1.5. mu.g/. mu.L total protein. The sample was boiled for 3 minutes, cooled and centrifuged. 15-20 μ g of protein from each sample was loaded onto a preformed 10-20% gradient Tris-HCl Criterion gel. The gel was electrophoresed in 1 XTTris-glycine-SDS running buffer at 180-. Fixing the gel in 40% methanol/10% acetic acid for 30-60 min, and staining with colloidal Coomassie blue G-250; at a minimum, overnight, or up to 3 days. To remove background, the gel was destained with deionized water. The gel was developed using a GS 800 calibratededensitometer densitometer. Quantification was performed using Bio-Rad Quantity OneSoft software. The software was used to determine the relative amount of each band in the sample lane. The% glycinin subunit and% β -conglycinin subunit are reported as relative percentages of total protein in the lanes.

Analysis of total amino acids: samples were assayed by three methods to obtain a complete profile (fulllprofile). Tryptophan requires alkaline hydrolysis with sodium hydroxide. Sulfur-containing amino acids require oxidation with performic acid before hydrolysis with hydrochloric acid. Analysis of the remaining amino acids of the sample can be accomplished by direct acid hydrolysis with hydrochloric acid. Once hydrolyzed, each amino acid was quantified using an automated amino acid analyzer (AOAC Official Methods of analysis of AOAC International, 2000).

Ash content: the sample was placed in a 550 ℃ electric furnace and allowed to burn to remove all volatile organic materials. The remaining nonvolatiles were quantified gravimetrically and calculated to determine the percent ash. AOAC official analytical methods, (2000).

Carbohydrate: the total carbohydrate level was calculated by subtraction using fresh weight derived data and the following equation: carbohydrate% 100% - (% protein +% fat +% moisture +% ash). Department of agriculture of the United states (1973).

Soxhlet extraction fat: the sample was weighed into a cellulose thimble containing sand or sodium sulfate and dried to remove excess moisture. The sample was saturated with pentane to extract the fat. The extract was then evaporated, dried and weighed. AOAC official analytical methods, (2000).

Moisture content: the sample was dried in a vacuum oven at about 100 ℃ to constant weight. The moisture weight loss was determined and converted to percent moisture.

Protein: the nitrogen-containing compounds in the sample are reduced in the presence of a boiling sulfuric acid and mercury catalyst mixture to form ammonia. Rendering the acid digest alkaline. The ammonia was distilled off and then titrated with a standard acid. The percent nitrogen was calculated and converted to protein content by a factor of 6.25. AOAC official analytical methods, (2000). Bradstreet, (1965). Kalthoff and Sandell (1948).

As a result: populations of high β -conglycinin soybean lines selected from hybrids with soybeans having low odor-producing traits, as measured by the formation of hexanal, hexanol, 2, 4-decadienal, and 1-octen-3-ol (table 8), showed widely varying odor-producing characteristics. Commercial soybeans have β -conglycinin protein at about 22% of the total protein and soy globulin protein at about 38%, compared to soybeans in table 8, which have greater than 30% of the protein as β -conglycinin protein and less than 25% of the protein as glycinin protein. Soybeans were constructed with greater than 30% total protein as β -conglycinin, producing less than 20 μ g/g total hexanal plus hexanol and 2, 4-decadienal in the odor assay of example 1, and also containing low levels of free asparagine and free arginine (table 8). For example, the present invention creates 20 β -conglycinin lines that produce less than 20 μ g/g total 2, 4-decadienal plus hexanal plus hexanol, and total free arginine plus asparagine between 360-. These lines had between 35-1,000. mu.g/g ground soybean free asparagine and between 500-2400. mu.g/g ground soybean free arginine.

Tables 8A and B: the characteristics of soybean progeny that are harvested in the united states for high beta-conglycinin. Soybeans were constructed having greater than 30% total protein as beta-conglycinin and less than 25% protein as glycinin, and produced less than 20 μ g/g 2, 4-decadienal plus hexanal plus hexanol per gram of soybean flour in the odor assay of example 1. The lipoxygenase assay is not useful for identifying low odor producing strains.

TABLE 8A

Sample ID	Beta-conglycinin (% of total protein)	Hexanal (mu g/g)	Hexanol (μ g/g)	Octen-3-ol (μ g/g)	Decadienal (μ g/g)	Hexanal + HexOH (μ g/g)	Hexanal + HexOH +2, 4-decadienal (. mu.g/g)	Liposaccharidase Activity, pH7	Liposaccharidase Activity, pH9	Asn(μg/g)	Arg(μg/g)	Arg+Asn(μg/g)
													HiBCSoy52	38.4	5.4	4.3	1.5	3.2	9.7	12.9	7.734	5.731	2024	4421	6445
HiBCSoy70	39.5	6.1	5.3	6.1	2.8	11.4	14.2	6.483	11.347	771	2020	2791
													HiBCSoy41	43.2	5.6	6.1	9.1	2.8	11.7	14.5	8.626	8.499	72	994	1066
HiBCSoy65	39.2	6.2	5.4	5.4	3.2	11.6	14.8	8.393	13.535	1939	5064	7003
													HiBCSoy35	42.0	6.2	5.3	8.5	3.9	11.6	15.5	6.935	9.623	171	757	928
HiBCSoy67	40.4	6.7	5.5	2.6	3.4	12.2	15.6	7.019	8.377	1376	3323	4699
													HiBCSoy34	40.6	5.2	7.8	7.4	3.0	13.0	16.0	7.279	10.633	311	1382	1693
HiBCSoy64	39.3	7.0	6.2	3.3	2.7	13.3	16.0	6.622	8.044	1723	3241	4964
													HiBCSoy62	41.1	6.9	6.7	1.1	2.7	13.6	16.2	5.697	10.358	2882	3938	6820
HiBCSoy68	40.5	7.8	5.8	2.3	2.9	13.6	16.5	8.006	6.584	303	1655	1958
													HiBCSoy61	30.3	9.6	4.7	4.1	2.3	14.4	16.6	6.709	6.110	372	1664	2036
HiBCSoy69	39.9	7.7	5.0	3.5	4.0	12.7	16.7	7.836	7.668	1393	4151	5544
													HiBCSoy48	44.5	7.1	6.4	2.2	3.3	13.5	16.8	7.601	3.783	99	1496	1595
HiBCSoy40	45.0	5.7	6.7	6.9	4.4	12.4	16.9	7.893	8.897	956	1878	2834
													HiBCSoy11	48.2	7.5	4.0	8.0	5.6	11.5	17.1	6.447	13.771	107	1680	1787
HiBCSoy42	38.6	7.4	6.0	6.0	3.7	13.4	17.1	6.825	11.446	77	289	366
													HiBCSoy53	40.0	6.9	6.7	4.2	4.2	13.6	17.9	6.649	6.849	1154	2489	3643
HiBCSoy5	46.5	8.0	6.2	5.2	3.9	14.3	18.2	5.882	5.793	305	3109	3414

HiBCSoy36	40.6	8.0	5.0	10.4	5.2	13.1	18.3	9.259	9.062	38	536	574
													HiBCSoy3	46.8	6.7	6.3	5.0	5.6	13.0	18.6	7.790	12.639	89	817	906
HiBCSoy4	44.9	7.4	6.7	8.6	4.6	14.1	18.6	6.929	8.660	723	3448	4171
													HiBCSoy46	42.4	6.7	8.5	9.4	3.5	15.1	18.7	6.978	3.850	67	1114	1181
HiBCSoy26	44.7	7.8	7.6	14.4	3.4	15.3	18.8	6.148	6.667	225	863	1088
													HiBCSoy18	44.8	9.3	4.6	7.6	5.0	13.9	18.9	9.482	7.439	121	504	625
HiBCSoy6	45.9	8.0	6.8	10.6	4.4	14.8	19.1	8.298	7.876	105	1149	1254
													HiBCSoy17	43.7	7.7	5.7	12.6	5.8	13.3	19.1	9.689	6.337	298	2363	2661
HiBCSoy44	31.7	7.6	7.2	7.2	4.7	14.8	19.4	8.909	6.160	125	569	694
													HiBCSoy63	33.5	10.3	6.7	1.9	2.7	16.9	19.6	6.177	10.239	677	1473	2150
HiBCSoy57	36.9	8.3	5.8	6.0	5.6	14.0	19.6	8.831	13.258	1329	3515	4844
													HiBCSoy16	44.3	8.5	6.1	10.8	5.3	14.6	19.9	8.326	6.447	95	863	958
HiBCSoy24	43.7	6.5	9.3	6.2	4.2	15.8	20.0	7.293	9.330	1083	1909	2992
													HiBCSoy58	36.4	7.7	7.3	3.2	5.0	15.1	20.1	8.078	14.134	1519	4252	5771
HiBCSoy39	41.7	7.2	6.5	4.5	6.3	13.7	20.1	9.084	5.718	95	400	495
													HiBCSoy66	39.2	8.8	7.4	2.6	4.2	16.2	20.4	6.122	9.155	2573	4163	6736
HiBCSoy38	37.6	7.9	5.7	5.3	7.0	13.6	20.6	9.356	9.776	71	592	663
													HiBCSoy59	37.2	9.6	5.2	5.0	5.8	14.8	20.6	10.126	8.492	271	1837	2108
HiBCSoy37	36.8	9.1	6.7	4.2	4.9	15.8	20.7	8.990	6.942	276	572	848
													HiBCSoy2	45.9	11.5	5.2	3.1	5.0	16.7	21.8	8.604	8.144	103	1116	1219
HiBCSoy60	40.1	9.6	6.1	7.8	6.1	15.7	21.8	7.463	7.468	1671	4330	6001
													HiBCSoy43	37.3	7.9	9.7	6.2	4.6	17.6	22.1	5.111	6.575	209	1083	1292
HiBCSoy25	44.2	10.0	8.5	8.3	3.9	18.4	22.3	6.642	7.495	686	1666	2352
													HiBCSoy50	46.3	8.8	6.0	6.2	7.6	14.8	22.3	7.072	6.590	115	1339	1454
HiBCSoy13	45.4	11.4	5.3	2.8	5.9	16.7	22.6	7.421	8.778	508	2290	2798
													HiBCSoy55	37.8	9.2	7.8	4.5	6.1	17.1	23.1	6.848	6.503	1705	4929	6634

HiBCSoy54	31.2	9.9	6.4	2.8	6.9	16.3	23.2	5.994	5.363	752	1569	2321
													HiBCSoy19	44.3	12.5	5.7	9.3	5.1	18.2	23.3	9.434	11.218	108	1257	1365
HiBCSoy14	46.1	8.8	6.5	8.6	8.2	15.3	23.5	7.974	7.297	181	1334	1515
													HiBCSoy20	41.4	10.2	8.9	10.4	4.6	19.1	23.7	6.392	10.686	1007	2329	3336
HiBCSoy45	45.7	9.1	9.3	3.8	5.5	18.4	23.9	7.637	15.149	169	744	913
													HiBCSoy12	48.6	10.8	7.6	4.9	6.1	18.4	24.5	9.695	10.837	92	455	547
HiBCSoy31	42.0	12.5	6.9	11.0	5.4	19.4	24.8	12.072	6.823	66	1162	1228
													HiBCSoy7	46.5	12.5	6.0	3.8	6.5	18.5	25.0	8.042	9.921	476	1641	2117
HiBCSoy1	45.1	12.3	6.7	6.2	6.5	19.0	25.6	8.351	7.376	187	1423	1610
													HiBCSoy29	38.8	4.0	10.1	8.4	11.6	14.1	25.7	8.944	5.006	136	818	954
HiBCSoy8	48.7	11.6	8.6	8.2	5.6	20.2	25.8	6.888	8.407	321	2612	2933
													HiBCSoy56	36.4	11.5	7.7	7.5	6.9	19.2	26.1	7.312	8.601	1155	2602	3757
HiBCSoy51	40.4	11.5	6.0	4.2	8.7	17.5	26.2	7.622	10.635	1695	3701	5396
													HiBCSoy47	38.1	10.0	11.6	6.4	4.8	21.6	26.4	6.974	4.039	92	1013	1105
HiBCSoy30	36.6	9.9	11.6	3.0	5.1	21.4	26.6	11.349	4.735	158	812	970
													HiBCSoy27	38.6	11.0	12.1	8.0	4.1	23.1	27.2	7.118	4.131	633	1082	1715
HiBCSoy28	38.8	12.7	9.6	6.9	6.3	22.2	28.6	4.762	5.822	2575	3204	5779
													HiBCSoy15	45.8	15.5	6.6	2.2	6.6	22.1	28.6	9.056	9.945	129	620	749
HiBCSoy23	43.7	13.0	10.2	10.0	6.1	23.2	29.4	6.681	5.207	1136	2578	3714
													HiBCSoy22	29.7	9.3	11.0	8.9	9.5	20.4	29.9	6.645	8.996	192	844	1036
HiBCSoy33	41.3	16.7	7.6	6.0	7.8	24.2	32.0	8.162	9.203	576	2919	3495
													HiBCSoy10	44.3	15.6	8.2	5.6	8.2	23.8	32.0	8.624	11.794	106	1108	1214
HiBCSoy21	35.7	14.5	12.0	10.8	6.1	26.5	32.6	8.634	11.921	99	581	680
													HiBCSoy9	46.9	16.6	8.7	5.9	8.7	25.2	33.9	6.976	6.340	155	1430	1585

TABLE 8B

Sample ID	Glycinin protein A1a, A1b, A2 and A4 (in% of total (%, dry basis) protein)	A3 glycinin	B1a, B1B, B2, B3, B4 glycinin	Alpha beta-conglycinin	Alpha' beta-conglycinin	Beta-conglycinin	Total beta-conglycinin	Total glycinin
									HiBCSoy52	42.76 4.872	4.784	9.097	20.849	12.381	5.123	38.353	18.753
HiBCSoy70	41.78 4.179	3.563	7.039	22.621	11.596	5.262	39.479	14.781
									HiBCSoy41	40.49	5.784	1.555	23.558	14.185	5.502	43.245	7.339
HiBCSoy65	43.82 4.759	5.007	7.737	22.044	12.875	4.263	39.182	17.503
									HiBCSoy35	40.56 2.312	4.538	4.913	22.903	12.727	6.415	42.045	11.763
HiBCSoy67	42.46 4.167	3.608	8.694	23.331	11.983	5.038	40.352	16.469
									HiBCSoy34	41.31 3.352	4.615	4.318	22.04	12.555	5.961	40.556	12.285
HiBCSoy64	43.10 4.949	5.047	7.773	21.646	12.79	4.858	39.294	17.769
									HiBCSoy62	44.64 4.891	5.045	6.098	22.73	12.045	6.309	41.084	16.034
HiBCSoy68	42.56 3.849	3.209	7.343	22.829	11.403	6.297	40.529	14.401
									HiBCSoy61	38.94 4.274	4.172	10.014	17.347	9.747	3.206	30.3	18.46
HiBCSoy69	42.64 4.468	3.81	8.614	22.386	11.791	5.713	39.89	16.892
									HiBCSoy48	39.72	6.168	2.907	24.062	15.07	5.414	44.546	9.075
HiBCSoy40	41.18	6.068	1.264	23.629	14.878	6.525	45.032	7.332
									HiBCSoy11	44.68	6.886	3.09	24.802	13.863	9.552	48.217	9.976
HiBCSoy42	38.13 1.822	4.93	3.401	20.961	11.577	6.06	38.598	10.153
									HiBCSoy53	43.66 4.782	4.783	8.585	22.103	12.521	5.373	39.997	18.15
HiBCSoy5	45.03	5.869	2.944	24.794	13.653	8.089	46.536	8.813
									HiBCSoy36	38.50 2.463	4.221	3.665	22.435	11.65	6.532	40.617	10.349
HiBCSoy3	42.55	6.308	3.171	25.442	14.402	6.927	46.771	9.479
									HiBCSoy4	43.56	5.449	2.765	23.395	14.297	7.2	44.892	8.214
HiBCSoy46	39.89	6.149	1.453	23.323	14.892	4.155	42.37	7.602

HiBCSoy26	41.21	5.742	2.31	23.643	14.391	6.636	44.67	8.052
									HiBCSoy18	40.14	5.385	1.875	23.836	14.999	5.934	44.769	7.26
HiBCSoy6	42.40	5.598	2.769	24.62	13.114	8.123	45.857	8.367
									HiBCSoy17	39.74	5.788	2.281	22.884	13.984	6.816	43.684	8.069
HiBCSoy44	39.01 4.766	4.241	8.322	17.716	9.373	4.63	31.719	17.329
									HiBCSoy63	41.95 3.372	3.558	11.179	17.431	10.613	5.467	33.511	18.109
HiBCSoy57	43.94 5.167	5.105	8.459	21.038	11.288	4.558	36.884	18.731
									HiBCSoy16	40.83	5.284	2.94	23.351	14.266	6.694	44.311	8.224
HiBCSoy24	43.66 2.799	4.931	5.949	23.351	13.832	6.508	43.691	13.679
									HiBCSoy58	43.60 5.132	4.262	8.898	21.14	11.418	3.834	36.392	18.292
HiBCSoy39	37.61 2.666	5.814	5.48	21.436	13.648	6.63	41.714	13.96
									HiBCSoy66	44.77 5.063	4.755	9.443	21.266	12.448	5.5	39.214	19.261
HiBCSoy38	38.42 3.923	3.871	5.194	20.716	11.702	5.154	37.572	12.988
									HiBCSoy59	40.54 4.696	4.26	7.023	21.554	12.26	3.384	37.198	15.979
HiBCSoy37	38.99 4.581	4.392	6.76	19.84	10.994	5.954	36.788	15.733
									HiBCSoy2	41.47	5.872	2.553	25.298	13.934	6.684	45.916	8.425
HiBCSoy60	43.40 5.019	4.688	7.532	22.035	12.045	5.976	40.056	17.239
									HiBCSoy43	43.12 4.856	4.538	7.757	20.84	12.396	4.097	37.333	17.151
HiBCSoy25	42.23	5.976	3.074	23.404	14.578	6.257	44.239	9.05
									HiBCSoy50	41.44	6.019	1.903	25.113	16.165	4.972	46.25	7.922
HiBCSoy13	45.51	5.732	2.562	24.536	13.744	7.135	45.415	8.294
									HiBCSoy55	43.70 5.036	4.671	8.171	20.641	12.032	5.142	37.815	17.878
HiBCSoy54	42.9 3.604	3.3	12.991	16.118	10.006	5.063	31.187	19.895
									HiBCSoy19	41.60	6.109	2.67	22.8	14.199	7.284	44.283	8.779
HiBCSoy14	42.22	5.431	2.557	24.628	14.439	7.002	46.069	7.988
									HiBCSoy20	41.04 1.577	4.913	4.422	21.592	13.422	6.411	41.425	10.912

HiBCSoy45	40.13	5.347	2.737	24.384	14.056	7.308	45.748	8.084
									HiBCSoy12	39.59	5.842	3.582	26.33	15.218	7.082	48.63	9.424
HiBCSoy31	40.25 1.7	4.33	2.41	22.579	13.279	6.133	41.991	8.44
									HiBCSoy7	42.12	5.905	2.788	25.149	13.985	7.401	46.535	8.693
HiBCSoy1	40.11	5.451	2.366	23.373	13.84	7.865	45.078	7.817
									HiBCSoy29	40.01 3.919	3.571	6.9	20.207	13.234	5.345	38.786	14.39
HiBCSoy8	46.86	6.055	3.495	25.584	14.699	8.464	48.747	9.55
									HiBCSoy56	42.14 4.915	4.981	8.294	21.474	12.173	2.736	36.383	18.19
HiBCSoy51	42.24 4.303	4.695	7.503	22.456	13.402	4.535	40.393	16.501
									HiBCSoy47	39.32	5.83	1.61	20.486	11.803	5.835	38.124	7.44
HiBCSoy30	43.28 4.796	4.07	8.302	19.29	11.788	5.555	36.633	17.168
									HiBCSoy27	43.71 4.651	4.769	7.697	20.396	12.062	6.173	38.631	17.117
HiBCSoy28	44.14 5.017	4.986	8.742	20.351	12.578	5.915	38.844	18.745
									HiBCSoy15	42.65	6.124	2.44	23.224	14.71	7.892	45.826	8.564
HiBCSoy23	41.51 2.38	5.072	5.094	23.148	13.627	6.932	43.707	12.546
									HiBCSoy22	42.24 5.253	4.672	9.326	14.996	9.178	5.509	29.683	19.251
HiBCSoy33	41.71 2.126	5.273	4.404	22.994	13.025	5.292	41.311	11.803
									HiBCSoy10	41.40	5.364	2.383	23.984	15.345	4.929	44.258	7.747
HiBCSoy21	39.50 3.867	3.566	6.852	18.347	11.398	5.944	35.689	14.285
									HiBCSoy9	42.92	6.2	2.45	24.453	14.776	7.647	46.876	8.65

The lipoxygenase activity of each high β -conglycinin soybean sample was very different. The lipoxygenase activity at pH7 and pH9 of the same soybean meal was independent of the total amount of 2, 4-decadienal plus hexanal plus hexanol produced (R)²The value was < 0.02).

Another experiment was conducted to demonstrate that soybeans with greater than 30% beta-conglycinin and less than 25% glycinin could be selected for low color, with amino acid composition in the range of commercial soybeans. Four high β -conglycinin soybeans were selected that also contained the Roundup Ready ® trait at yields equal to or greater than the average yield of the commercial standard line at three locations. Average b of about 22^*And L averaging about 85^*(Table 9) these soybeans demonstrated low color traits. The amino acid composition (table 9) falls within the commercial soybean range (table 10) published in the International Life Sciences Institute Crop composition database (International Life Sciences Institute Crop composition database, version 1.0, 3 months and 22 days of 2004).The average amino acid composition of the three high β -conglycinin soybean lines (table 9) was compared to the average soybean composition in the ILSI database (table 10). The four amino acids (arginine, lysine, histidine and serine) of the high β -conglycinin line differ from the average composition of commercial soybeans by between 10-15% but are still within the range of commercial soybean compositions.

It is also desirable to hybridize high beta-conglycinin soybeans with low linolenic acid content, medium oleic acid content soybeans to further improve their organoleptic properties. Soybeans with low linolenic acid content and medium oleic acid content were constructed using traditional breeding methods at Monsanto, and contained about 2% linolenic acid content, about 25% linoleic acid content, and about 59% oleic acid content in total fatty acids.

Table 9: composition, color and flavor properties of soybeans cultivated in three places in 2003. All soybeans had the Roundup Ready ® trait and dark hilum.

	HBC-360			HBC-350			HBC-390
											Mean value of	stdev		Mean value of	stdev		Mean value of	stdev
B-conglycinin (% of total protein)	37.4	1.5		37.0	0.3		37.9	1.0
										Glycinin (in% of total protein)	15.4	0.8		15.2	1.3		17.1	0.5
Free Asn (ppm)	307	224		183	124		535	309
										Free Arg (ppm)	2825	1211		2050	829		2791	674
										Colour(s)
b*	22.0	1.6		23.3	1.9		22.7	0.6
										L*	85.4	0.4		85.6	0.7		84.8	0.1
										Smell(s)
Hexanal + hexanol +2, 4-decadienal (. mu.g/g soybean)	25.1	1.2		53.7	17.0		29.4	1.0
Approximate values (g/100g soybean)
										Moisture content	5.91	0.06		5.855	0.09		5.97	0.11
Protein	37.40	1.15		36.05	0.49		37.87	1.62
										Total fat	17.53	0.25		18.2	0.42		16.27	0.15
Ash content	4.41	0.28		4.45	0.18		4.36	0.14
										Carbohydrate compound	34.77	0.80		35.45	0.21		35.57	1.59

										Amino acids	mg/g Soybean		mg/g protein	mg/g Soybean		mg/g protein	mg/g Soybean		mg/g protein
Aspartic acid	39.90	0.95	106.68	40.05	0.35	111.10	42.50	1.01	112.24
										Threonine	12.70	0.60	33.96	12.3	1.70	34.12	12.37	0.21	32.66
Serine	19.70	0.72	52.67	20.25	1.20	56.17	21.40	0.46	56.51
										Glutamic acid	62.67	1.79	167.56	62.7	0.42	173.93	67.13	1.16	177.29
Proline	18.33	0.38	49.02	18.35	0.07	50.90	18.67	0.49	49.30
										Glycine	15.27	0.32	40.82	15.2	0.00	42.16	15.80	0.26	41.73
Alanine	15.57	0.32	41.62	15.6	0.00	43.27	15.90	0.20	41.99
										Cystine	5.31	0.34	14.21	5.915	0.06	16.41	5.81	0.33	15.33
Valine	17.13	0.72	45.81	17.1	0.14	47.43	17.63	0.25	46.57
										Methionine	4.45	0.47	11.89	4.85	0.06	13.45	4.83	0.11	12.75
Isoleucine	16.33	0.55	43.67	16.4	0.14	45.49	16.67	0.25	44.01
										Leucine	27.80	0.78	74.33	27.95	0.07	77.53	28.93	0.25	76.41
Tyrosine	12.33	0.15	32.98	12.55	0.07	34.81	12.90	0.53	34.07
										Phenylalanine	18.37	0.61	49.11	18.4	0.00	51.04	18.83	0.31	49.74
Histidine	10.53	0.35	28.16	10.5	0.14	29.13	11.17	0.31	29.49
										Lysine	25.07	0.93	67.02	24.85	0.07	68.93	26.13	0.80	69.01
Arginine	27.03	1.45	72.28	26.45	0.49	73.37	30.17	1.75	79.67
										Tryptophan	3.96	0.11	10.58	3.945	0.02	10.94	4.20	0.17	11.08
Met+Cys			26.10			29.86			28.08

Table 10: comparison of the average composition of the three high β -conglycinin soybeans with that of the crop composition database (version 1.0) of the international institute of life sciences. Selection criteria to obtain data: crop type soybean-glycine max, tissue type: seed, year of farming: all, country: all, state: all. Abbreviations: FW is soybean meal weight and DW is dry weight.

Analyte	Minimum value	Maximum value	Mean value of	n	Unit of	Average HBC Soybean (mg/g FW)	Difference from ILSI average (%)
								Amino acid-alanine	13.6	17.2	15.35	187	mg/g FW	15.7	2.2
Amino acid arginine	20.9	31.3	25.31	187	mg/g FW	27.9	10.2
								Amino acid aspartic acid	33.9	46.6	40.16	187	mg/g FW	40.8	1.6
Amino acid cystine/cysteine	3.43	7.29	5.26	187	mg/g FW	5.7	8.0
								Amino acid glutamic acid	52.8	74	63.24	187	mg/g FW	64.2	1.5
Amino acid-glycine	13.1	17.4	15.03	187	mg/g FW	15.4	2.6
								Amino acid-histidine	8.07	10.8	9.32	187	mg/g FW	10.7	15.2
Amino acid-isoleucine	13.3	19	16.19	187	mg/g FW	16.5	1.7
								Amino acid leucine	23.1	31.5	27.04	187	mg/g FW	28.2	4.4
Amino acid-lysine	20	26.6	22.88	187	mg/g FW	25.4	10.8
								Amino acid-methionine	3.99	6.14	4.92	187	mg/g FW	4.7	-4.3
Amino acid phenylalanine	14.6	20.5	17.69	187	mg/g FW	18.5	4.8
								Amino acid-proline	15.1	21.2	18.05	187	mg/g FW	18.5	2.2
Amino acid-serine	15	22.8	18.12	187	mg/g FW	20.5	12.9
								Amino acid-threonine	11.5	15.1	12.99	187	mg/gFW	12.5	-4.1

Amino acid-tryptophan	3.26	4.7	3.903	187	mg/g FW	4.0	3.3
								Amino acid-tyrosine	8.7	14.3	11.78	187	mg/g FW	12.6	6.9
Amino acid valine	14.5	20.5	17.14	187	mg/g FW	17.3	0.9
								Analyte	Minimum value	Maximum value	Mean value of	n	Unit of
Near-ash content	3.885	6.542	5.313	237	％DW
						Approximate-calculated carbohydrates	29.6	50.2	38.1	237	％DW
Approximate-crude protein	33.19	45.48	39.28	237	％DW
						Approximate-total fat	8.104	23.562	16.94	237	％DW

Example 5

Comparison of odor and color traits of commercial soybeans with soybeans selected according to the present invention

The odor and color characteristics of commercial soybeans were determined for comparison with those of the soybeans of the present invention. In the odor assay, some soybeans produced less than 17.5. mu.g/g hexanal plus hexanol, and some produced less than 11. mu.g/g 2, 4-decadienal (Table 11). However, none of the commercial soybeans produced less than 20 μ g of total 2, 4-decadienal plus hexanal plus hexanol per gram of ground seeds after oxidation under mild aqueous conditions. Four lipoxygenase lines of the invention were constructed which produced less than 20. mu.g/g hexanal + hexanol +2, 4-decadienal in two seasons (tables 4, 5). For example, line A-1 harvested in 2001 and 2002 yields a total of hexanal + hexanol and 2, 4-decadienal of 8.0 and 18.2. mu.g/g, respectively (tables 4, 5). The present invention created more than 20 high β -conglycinin lines producing less than 20 μ g/g total hexanal + hexanol and 2, 4-decadienal (table 8). For example, a high β -conglycinin soybean produced 9.7 μ g/g hexanal + hexanol and 3.2 μ g/g 2, 4-decadienal (Table 8).

Commercial soybeans are very wide in color range. E.g. b^*The values ranged from 27-34 (Table 11). It was found that the soybean line of the present invention can be used as^*The values extend to 22 (table 4, table 9).

Tables 11A and B: color and odor generating characteristics of harvested commodity soybeans

TABLE 11A odor

Sample numbering	Hexanal (pg/g)	Hexanal (pg/g)	Hexanol (pg/g)	2, 4-decadienal	1-Octen-3-ol (MS' S)	2, 4-decadienal + hexanal + hexanol (μ g/g)
							385493491-2210287387138379137209386390	14.05.05.015.414.015.614.917.415.015.515.718.1	3.95.14.45.13.63.95.04.14.85.53.93.9	18.010.19.420.517.619.619.921.519.921.019.722.0	2.811.114.33.67.25.35.03.45.14.25.63.7	1.85.46.17.018.48.59.39.18.43.15.711.2	20.721.223.724.124.824.824.924.925.025.225.325.8

Sample numbering	Hexanal (pg/g)	Hexanal (pg/g)	Hexanol (pg/g)	2, 4-decadienal	1-Octen-3-ol (MS' S)	2, 4-decadienal + hexanal + hexanol (μ g/g)
							285286290289175383411276378282224139492216381384288280557206-2141271299208495284211486215142485271-2494388444291377-2140249293292275	13.914.814.514.89.516.75.814.717.715.514.517.04.916.119.318.515.917.418.917.617.615.915.817.95.018.61854.916.416.75.115.35.118.512.015.817.116.518.215.515.517.3	6.25.35.55.85.83.97.95.93.96.15.35.24.55.84.14.15.26.16.15.15.67.04.85.15.66.35.93.36.15.35.37.36.24.05.66.43.86.55.15.05.87.5	20.120.120.020.615.320.613.820.521.621.719.822.29.321.823.422.621.123.525.022.823.322.920.623.010.524.924.48.222.522.010.322.611.222.617.622.220.922.923.920.421.324.8	596.06.66.111.66.613.57.26.26.18.16.019.06.75.26.07.55.23.65.95.55.88.36.118.54.24.721.06.77.218.96.718.16.911.97.48.7696.09.68.75.3	4.612.36.47.84.37.98.02.59.65.82.17.34.52.29.712.41.43.75.76.09.13.32.74.85.81.74.36.72.513.15.34.03.77.92.62.24.63.75.26.52.44.4	26.026.126.626.826.927.227.327.727.827.828.028.128.328.528.528.628.728.728.728.728.728.729.029.029.129.129.129.129.229.229.229.329.429.429.529.629.629.929.930.030.130.1

Sample numbering	Hexanal (pg/g)	Hexanal (pg/g)	Hexanol (pg/g)	2, 4-decadienal	1-Octen-3-ol (MS' S)	2, 4-decadienal + hexanal + hexanol (μ g/g)
							274296260213295570235490294481380206377382481-22622793002272982312204004821252255392122813619255141654727724826723211245401255	16.413.916.217.315.417.616.25.11754.920.117.818.621.35.116.918.116.816.914.918.617.47.55.116.718.020.417.017.217.110.420.37.320.818.720.717.016.516.018.321.519.2	6.76.65.84.95.76.84.85.06.14.24.15.23.84.01.95.68.15.96.06.06.47.09.16.74.45.85.83.88.05.16.58.07.06.46.45.75.66.16.46.85.97.2	23.120.622.022.221.224.421.010.123.79.124.222.922.425.27.022.426.222.822.920.925.024.416.611.721.123.826.220.825.222.216.928.214.327.225.126.422.522.622.425.027.526.4	7.19.88.48.39.56.39.820.87.221.86.88.18.66.024.59.05.38.88.610.76.87.415.420.210.98.35.911.37.010.015.64.318.45.6196110.610.510.78.15.77.1	5.73.04.92.55.64.17.25.74.34.37.25.95.65.65.04.82.8621.93.89.04.06.84.32.20.63.88.86.60.05.44.46.34.15.36.54.93.02.61.58.95.1	30.230.430.430.530.730.730.830.930.930.931.031.131.131.331.431.531.531.531.531.631831.932.032.032.032.132.132.132.232.332.532.532.732.832.933.033.133.133.133.133.133.5

Sample numbering	Hexanal (pg/g)	Hexanal (pg/g)	Hexanol (pg/g)	2, 4-decadienal	1-Octen-3-ol (MS' S)	2, 4-decadienal + hexanal + hexanol (μ g/g)
							230283349166272483236491571376-2147148266242222-225022254941219278149233185389221348136-2218217256555346489228261189566404114136102	17.720.411.48.319.55.119.35.121.021.120.719.418.620.118.520.618.321.220.719.019520.219414.224.621.419.220.019.119.118.424.219.85.120.618.214.122.122.722.520.018.8	6.35.98.08.77.52.06.34.67.33.95.56.06.15.65.46.25.38.66.26.47.55.46.85.54.15.35.74.86.15.76.17.46.54.46.27.06.07.86.03.24.85.3	24.026.319.417.027.17.125.69.628.325.026.125.424.625.823.926.823.629.726.925.427.025.626.219.728.726.724.824.825.224.824.531526.29.526.825.320.129.928.725.724.824.0	9.47.314.416.86.826.98.424.45.89.18.19.210.09.110.98.111.35.38.39.88.39.79215.76.88.710.710.810.511.111.44.59.726.69.310.916.26.47.911.011.912.8	2.81.12.44.10.83.50.46.93.05.713.98.60.52.85.38.55.23.94.0622.711.21.45.52.35.83.611.57.116.66.05.33.15.47.21.112.70.84.910.714.65.8	33.533.633.833.833.933.934.034.034.134.134.234.734.734.934.934.934.935.135.235.235.235.335.435.435.435.535.535.635.835.936.036.036.036.136.136.236.336.336.536.736.736.8

Sample numbering	Hexanal (pg/g)	Hexanal (pg/g)	Hexanol (pg/g)	2, 4-decadienal	1-Octen-3-ol (MS' S)	2, 4-decadienal + hexanal + hexanol (μ g/g)
							3763522235444054548855918O41714342848735830517356140525920229727039177223-2360273286-2440229550399236-224745484558432556-2554198546	21.917.519.422.622.025.45.120.918.65.521.45.15.113.320.713.825.724.819.217.118.120.123.017.021.114.022.327.117.122.626.68.022.921.823.15.122.513.525.822.018.726.8	4.16.95.99.65.67.55.26.96.58.15.88.55.37.56.86.48.26.26.85.68.05.65.96.46.17.36.66.36.98.37.110.46.27.06.26.17.516.06.08.65.57.1	26.024.425.432.227.632.910.327.825.113.627.113.510.320.827.520.334.031.126.122.726.025.728.823.527.221.329.033.524.030.833.718.429.028.729.311.230.029.531.730.624.233.9	10.912.811.95.19.84.527.29.712.524.110.624.227.517.110.417.84.17.112.215.612.312.79.615.011.417.39.95.414.98.15.520.810.310.710.128.59.710.38.19.415.96.2	6.33.43.15.10.92.77.04.99.87.616.08.36.64.25.92.35.41.94.24.72.15.05.76.53.53.17.326.11.00.37.34.60.88.65.72.13.56.17.93.95.84.8	36.937.237.237.337.437.437.537.537.537.737.737.837.837.937.938.138.138.138.338.338.338.438.438.538.538.638.838.838.938.939.239.239.339.439.439.739.739.839.940.040.140.1

Sample numbering	Hexanal (pg/g)	Hexanal (pg/g)	Hexanol (pg/g)	2, 4-decadienal	1-Octen-3-ol (MS' S)	2, 4-decadienal + hexanal + hexanol (μ g/g)
							560272-2214425692738565108540174342-2315538237-2310192-235439114641826357320034739536-223725122335256-2375203254563572536144241191461	22.623.220.723.622.021.822.528.020.726.020.622.912.027.523.520.519.618.923.522.415.922.122.717.921.325.024.123.128.222.816.322.323.919.025.822.725.126.824.027.114.426.0	7.27.86.96.17.35.85.07.24.69.26.07.15.68.O6.07.45.110.73.85.67.05.88.75.77.36.15.16.37.65.79.17.13.95.56.47.67.85.95.05.56.51.3	29.831.027.529.629.327.727.535.225.335.126.730.017.635.529.528.024.729.627.328.023.027.931.423.628.631.029.229.435.728.525.429.427.824.532.230.333.032.729.132.620.927.3	10.39.113.011.011.312.913.25.515.55.714.311.023.55.611.713.216.611.814.113.618.613.710.418.213.711.413.313.16.814.117.213.214.918.110.512.69.910.213.910.422.115.7	4.61.03.53.54.95.88.05.95.52.58.01.65.35.68.08.35.95.07.19.35.74.03.94.75.19.20.49.16.05.54.39.44.96.23.22.92.36.61566.54.36.8	40.240.240.540.640.640.640.740.740.840.841.041.041.141.141.241.241.341.341.441.641.641.741.841.842.342.442.542.542.542.642.642.642.742.742.842.942..942.943.043.043.043.0

Sample numbering	Hexanal (pg/g)	Hexanal (pg/g)	Hexanol (pg/g)	2, 4-decadienal	1-Octen-3-ol (MS' S)	2, 4-decadienal + hexanal + hexanol (μ g/g)
							359179556541-2252201257269301191-2190407562178244445151111-2145542531246153725844620519711272240460153534447571323323181127172	22.620.026.825.625.118.625.123.622.818.922.614.825.122.529.726.424.816.225.123.728.422.425.027.023.522.220.920.824.924.727.526.725.522.626.725.924.422.419.823.224.622.5	7.45.43.86.69.06.35.46.25.96.65.77.17.87.96.76.75.413.56.76.45.46.15.65.96.07.05.45.9516.47.31.85.37.87.06.55.16.85.55.45.21.9	30.025.430.632.234.124.830.429.828.725.528.421.932.930.436.433.130.329.831.830.133.928.530.633.029.529.226.426.730.631.134.828.530.830.333.832.430.129.225.328.529.831.4	13.017.712.611.09.218.613.414.115.218.415.622.111.213.87.811.113.914.412.414.210.616.013.911.615.215.518.418.114.313.910.316.614.314.911.613.015.416.220.217.116.014.4	4.910.27.46.20.55.83.69.03.24.34.47.44.212.84.70.813.47.516.61.77.53.53.95.91.56.55.35.69.714.16.93.65.04.24.84.99.12.23.26.28.45.2	43.143.143.143.243.443.443.843.943.943.943.944.044.144.144.244.244.244.344.344.344.544.544.544.644.644.744.844.845.045.045.145.145.145.245.345.445.545.545.545.645.745.8

Sample numbering	Hexanal (pg/g)	Hexanal (pg/g)	Hexanol (pg/g)	2, 4-decadienal	1-Octen-3-ol (MS' S)	2, 4-decadienal + hexanal + hexanol (μ g/g)
							43681504515413121105485306615722634315216437433617156790350131345436309181-28119615470119419195396109347-25295030478121-262-2	26.823.624.521.926.915.324.928.729.024.627.031.222.925.825.025.126.921.031.926.821.126.921.922.324.623.128.322.627.525.528.713.419.925.626.125.424.228.322.526.325.727.1	6.67.16.22.76.87.33.97.47.37.05.77.28.25.16.64.06.313.09.66.19.25.48.36.57.65.95.05.15.35.63.88.86.05.65.47.87.57.56.46.75.75.9	33.430.730.724.633.722.628.826.236.331.732.838.431.030.931.629.133.234.041.532.830.332.330.228.832.228.933.327.732.831.132.622.225.931.331.533.131.735.829.033.131.432.9	12.515.315.321.412.523.617.310.110.114.813.88.115.615.815.217.713.612.95.414.116.614.716.918.314.818.213.919.514.516.314.925.321.716.316.114.616.112.219.115.116.715.2	0.915.79.64.55.15.310.76.45.39.89.34.96.911.2933.04.64.93.58.28.07.06.36.32.88.74.76.67.48.94.91.65.05.77.15.65.56.71.66.214.814.0	45.946.046.046.046.146.146.146346546.546.546.546.746.746.846.846.846.946.946.947.047.047.147.147.147.147.247.247.347.447.547.547547.547.547.747.848.048.048.148.248.2

Sample numbering	Hexanal (pg/g)	Hexanal (pg/g)	Hexanol (pg/g)	2, 4-decadienal	1-Octen-3-ol (MS' S)	2, 4-decadienal + hexanal + hexanol (μ g/g)
							5535114131063031821217626526434485394268535194443340129152-2128564337159105311302346-262444212381583389235680568456409452331	25.726.319.225.922.924.122.622.727.826.523.621.424.527.836.121.821.723.726.627.827.027.629.828.625.725.623.028.027.028.327.225.128.028.428.126.728.031.325.127.526.424.2	9.72.78.84.25.45.55.95.67.28.08.011.46.28.57.25.96.88.15.35.24.67.56.25.46.06.16.06.76.07.37.76.35.51.56.17.66.710.50.67.61.18.1	35.429.128.030.128.329.628.528.335.034.531.632.830.736.343.327.728.531.832.033.031.535.136.034.031.731.728.934.733.035.534.931.433.535.934.234.234.841.725.835.127.632.3	12.919.220.418.320.218.920.120.313.714.317.316.118.212.85.821.520.917.617.516.618.014.513.715.718.118.121.015.217.014.515.318.916.814.516.316.516.19.125.115.823.418.7	3.23.85.812.81.94.54.55.20.91.84.45.18.41.35.18.30.84.712.911.215.33.43.315.215.96.27.13.39.72.95.53.910.63.18.27.08.37.490.17.720.45.6	48.348.348.448.448.548.548.548.648.748.848.848.949.049.149.149.249.449.449.449.549.549.649.649.749.849.949.949.949.950.050.250.250.350.450.550.750.950.950.950.951.051.1

Sample numbering	Hexanal (pg/g)	Hexanal (pg/g)	Hexanol (pg/g)	2, 4-decadienal	1-Octen-3-ol (MS' S)	2, 4-decadienal + hexanal + hexanol (μ g/g)
							65193365462204115441307391-26354346133302-2243170254502394485194381117116346410737131319910410333943937218731730635586436-291	27.829.326.921.923.731.123.426.526.528.135.831.529.924.630.226.528.632.629.424.132.626.331.029.829.026.729.928.825.323.325.729.125.727.227.515.026.823.730.930.826.531.0	7.86.94.00.65.96.27.66.35.66.28.46.25.16.47.68.15.97.87.413.45.16.66.16.15.80.84.74.16.25.36.93.89.16.23.822.25.96.67.26.36.36.4	35.626.230.928.529.637.331.032.932.134.444.137.735.031.037.934.634.540.436.837.437.832.937.735.934.827.534.632.932.128.632.532.934.833.931.437.232.730.338.137.132..837.4	15.815.320.623.222.214.721.019.220.117.88.114.517.221.214.517.817.912.215.915.314.919.915.217.218.525.818.720.421.324.820.920.618.719.622.416.621.123.515.816.821.316.7	9.05.24.395.06.23.75.85.57.614.53.64.910.17.37.511.86.85.94.72.22.69.05.512.715435.520.68.69.76.317.913.44.86.56.42.17.68.04.411.76.29.0	51.451.551.551.751.852.052.052.152.252.252.252.252.252352.352.452.452.652.752.752.752.852.953.153353.353.353.353.353.453.553.553.553.653.853.853.853.953.953954.054.1

Sample numbering	Hexanal (pg/g)	Hexanal (pg/g)	Hexanol (pg/g)	2, 4-decadienal	1-Octen-3-ol (MS' S)	2, 4-decadienal + hexanal + hexanol (μ g/g)
							61-277156511-2281137642744932420731612128126451-2135393130357314184151-2101331-233032535140332246315543539746518332145721316-234	28.129.731.527.934.732.230.732.826.726.636.229.029.232.630.027.230.127.930.224.326.029.032.531.527.630.928.727.739.129.826.832.732.028.829.226.529.435.031.232.928.533.6	7.87.05.42.86.05.66.57.17.38.18.76.15.56.35.60.86.06.56.118.16.75.55.64.07.88.69.76.78.18.10.75.77.46.47.01.08.19.40.76.36.17.1	35.936.736.930.740.737..837.239.934.034.744.935.234.738.935.628.036.134.536.342.432.634.538.235.535.539.538.434.347.137..827.538.439.535.236.327.537.544.431.839.234.640.7	18.217.517.323.813.816.817.414.820.920.510.420.220.616.519.927.619.721.419.613.523.321.517.920.620.616.617.821.99.118.628.918.117.221.620.629.519.512.925.518.222.916.9	15.43.35.95.07.915.79.15.04.70.90.26.811.011.57.815.39.214.24.18.39214.59.05.07.15.64.86.34.636.76.34.24.310.444.8124.015.42.47.99.4	54.154.254.254.554.554.654.654.754.955.255.355.455.455.455.555.655.855.855.955.955.956.056.156.156.156.156.256.256.256.556.556.556.656.856.956.957.057.357.457.457.557.6

Sample numbering	Hexanal (pg/g)	Hexanal (pg/g)	Hexanol (pg/g)	2, 4-decadienal	1-Octen-3-ol (MS' S)	2, 4-decadienal + hexanal + hexanol (μ g/g)
							6994317-24223661888747352734111734246747165369370421-2533354343643261231201163618424353122-252818621-241532811840246613	30.531.928.325.935.128.932.227.831.528.633.628.628.931.734.133.230.538.829.131.134.122.030.827.032.033.234.533.134.831.130.532.732.735.332.626.926.536.032.831.831.8	6.98.36.010.04.39.47.34.66.77.96.61.91.17.06.96.24.14.17.87.46.310.23.98.75.94.06.24.16.35.78.85.76.68.26.27.08.13.96.51.06.6	37.440.234.335.939.438.339.532.438.236.540.236.536.638.741.039.434.642.937.038.540.432.234.735.737.837.240.837.241.136.839.338.439.343.538.833.934.639.939.432.838.4	20.217.423.522.018.519.718.525.619.921.718.021.821.719.717.419.024.015.921.920.718.827.024.523.621.522.218.722.318.823.120.621.620.916.721.426.425.820.521.227.922.4	11.48.38.86.26.5927.310.74.13.78.74.311.712.03.212.912.34.25.35.76.48.64.62.911.420.28.72.31.53.45.917.55.36.23.25.85.97.14.638.58.2	57.657.657.857.951.958.058.058.058.158.258.258.258.358.458.458.558.658.858.959.259.259.259.259.359.459.459.459.559.959.960.060.160.260.260.260.360.460.460.560.760.8

Sample numbering	Hexanal (pg/g)	Hexanal (pg/g)	Hexanol (pg/g)	2, 4-decadienal	1-Octen-3-ol (MS' S)	2, 4-decadienal + hexanal + hexanol (μ g/g)
							442614554231694583621243322-2233225375318420161744826406-255251-2424881221603988932351242991-26443345336710437-2521472565	27.132.928.429.236.027.835.833.635.732.736.234.036.136.928.426.834.034.339.033.832.442.237.531.437.934.936.736.136.634.532.030.637.138.932.431.136.235.633.935.133.440.736.1	12.18.10.86.18.52.23.95.96.46.06.76.511.26.19.211.36.17.46.46.79.17.86.51.66.1626.18.08.57.33.17.36.711.88.50.84.09.38.23.62.79.86.0	39.241.029.235.344.530.039.739.542.138.642.940.547.343.037.638.140.141.745.440.541.550.044.139.144.641.242.944.145.041.835.137.943.750.740.931.940.344.942.138.736.150.642.1	21.820.332.126.117.031.622.122.419.923.319.221.815.019.525.024.522.621.117.522.621.713.919.824.919.423.021.520.419.923.229.927.321.614.724.533.625.421.724.527.930.616.324.8	2.615.730.63.87.85.45.211.97.26.22.39.71.78.24.77.88.212.22.55.77.44.14.07.68.313.011.815.711.40.911.81138.69.62.341.54.72.13.47.86.615.23.2	60.961.361.361.461.461.561.861.962.062.062.162.362.362.562.662.662.762.862.963.163.263.963.963.963.964.264.464.464.965.065.065265.365.465.465.565.766.566.666.666.866.866.9

Sample numbering	Hexanal (pg/g)	Hexanal (pg/g)	Hexanol (pg/g)	2, 4-decadienal	1-Octen-3-ol (MS' S)	2, 4-decadienal + hexanal + hexanol (μ g/g)
							315323291342526408523373407-27952447851547685165205034063634998414308514509234466-2101-2162361-233432752552544594978381-2467526-2469500	41.348.934.938.934.045.936.335.942.437.737.944.334.136.433.137.535.738.539.138.736.447.039.432.030.936.939.739.132.538.740.240.339.835.538.448.843.735.839.244.954.442.846.936.033.2	6.38.99.15.311.27.17.85.44.06.87.93.97.04.73.39.13.65.53.46.83.98.57.512.26.93.95.010.21.910.57.73.810.412.88.36.610.83.04.86.46.30.96.95.55.9	47.557.844.144.245.253.044.241.346.444.545.848.241.141.236.446.639.244.042.545.540.355.546.844.237.840.844.749.340.449.247.944.150.248.346.855.454.538.844.151.260.743.753.741.539.1	19.710.124.024.023.015.724.627.722.724.823.721.328.428.533.323.130.626.127.824.930.315.123.926.733.330.426.822.431.522.924.628.422.824.826.618.519.435.630.823.814.331.421.834.137.3	7.55.65.114.97.92.78.27.46.07.67.77.63.98.06.26.98.38.76.89.76.93.011.93.93.84.87.33.836.68.112.03.43.92.87.94.016.64.16.68.74.121.84.36.57.2	67.367.968.168.268.368.868.869.169.169.369.469.569.569.669.769.869.970.270.370.570.670.770.870.971.171.271.471.771.972.172.572.573.073.073.473.973.974.474.975.175.175.175.575.676.3

Sample numbering	Hexanal (pg/g)	Hexanal (pg/g)	Hexanol (pg/g)	2, 4-decadienal	1-Octen-3-ol (MS' S)	2, 4-decadienal + hexanal + hexanol (μ g/g)
							731005184375819467-2508454499333431167480650141047146847547050247747936860166-241293510474539953752211.25550432031950695	44.643.140.028.342.644.745.348.037.741.044.040.154.441.346.448.848.238.838.736.736.739.443.241.046.849.049.747.352.542.441.455.251.568.945.253.256.043.551.051.655.054.3	7.39.34.315.415.27.31.84.91.25.08.77.06.95.59.17.39.03.95.66.37.43.92.96.54.04.08.18.44.26.05.86.58.18.96.115.89.85.212.210.65.17.5	51.952.444.343.757.852.047.152.938.946.052.747.161.246.855.556.157.242.744.343.044.143.346.147.550.853.057.855.856.848.447.261.759.677.851.369.065.848.763.262.260.161.8	24.624.232.433.119.225.130.124.338.431.725.130.816.831.322.722.321.636.134.936.135.236.133.632.929.627.723.225.326.535.337.224.326.58.435.618.121.538.824.626.528.927.9	9.86.45.44.916.912.73.88.78.37.04.58.39.55.911.98.43.77.27.46.56.512.55.96.28.613.73.86.96.47.510.87.610.14.07.93.511.48.25.84.86.07.9	76.676.676.776.877.177.177.277.277.277.777.877.978.078.178.278.478.778.879.179.179.379.379.780.480.480.681.081.183.283.784.486.086.086.286.987.187.387.587.788.788.989.7

Sample numbering	Hexanal (pg/g)	Hexanal (pg/g)	Hexanol (pg/g)	2, 4-decadienal	1-Octen-3-ol (MS' S)	2, 4-decadienal + hexanal + hexanol (μ g/g)
							305179629496512 25971620513498914496 250797430125081739242560682 Range average value Stdev	57.459.153.561.260.653.455.856.660.467.264.360.367.465.879.178.169.276.174.183.377.181.792.190.286.9119.6119.94.9-12028.513.9	6.65.47.97.15.23.59.511.311.68.14.14.88.47.46.06.210.814.011.56.56.78.214.712.78.315.450.50.6-516.62.9	64.064.561.468.365.956.965.367.972.175.368.465.175.873.285.284.380.090.185.689.883.889.8106.8102.995.2135.0170.47.0-17035.015.1	25.925.728.926.129.638.630.528.729.326.837.241.432.135.828.429.534.925.130.629.936.036.620.528.841.324.113.92.8-4117.47.8	8.210.59.63.84.513.714.18.511.89.910.810.39.1784.56.511.23.72.97.87.610.115.08.225.84.19.00-957.37.0	89.990.390.394.495.595.595.996.6101.4102.1105.6106.5107.9109.0113.5113.8114.9115.2116.2119.6119.8126.5127.4131.7136.5159.1184.421-18452.420.1

Table 11B: color sample name ID: 47	b*27.18	L*82.34
			ID:42ID:45JA:48	27.2927.4027.88	83.0281.5884.41
LB:32	28.10	83.24

LB:33LB:30	28.2428.47	81.9980.80
			JA:45	28.62	82.83
LB:26JA:44	28.7128.86	82.4384.03
			ID:34	28.94	80.43
KA:45D:15	29.1729.43	84.2979.01
			D:48	29.56	79.52
LB:15ID:28	29.6030.05	82.4178.60
			KA:44	30.83	81.90
JB:12JB:16	31.1731.42	78.9979.02
			KA:25	32.69	80.71
LB:9JA:49	32.8933.48	79.9779.05
			JB:14	33.80	77.60
Range of	27-34	77-84
			Mean value of	29.73	81∶22

Example 6

Demonstration of the ability to combine low odor producing traits with low linolenic acid compositional traits

This example demonstrates that it is possible to combine low odor producing traits with low linolenic acid composition traits, and the effect of dehulling soybeans on odor production is further investigated. The percentage of linoleic acid in the soy fatty acids is typically about 8%. It was possible to construct soybeans containing less than 6% linolenic acid and having low odor-producing properties using traditional breeding methods (Table 12).

The formation of 1-octen-3-ol was measured independently of the formation of other volatile compounds in the odor assay. One hypothesis is that fungal enzymes on the surface of soybeans are a source of rapid 1-octen-3-ol formation in the assay. It was theorized that dehulling soybean seeds prior to grinding to flour should reduce the levels of fungal enzymes in the odor assay. Typically whole soybeans are ground to produce soybean flour for odor determination. Six carefully dehulled seeds of the low odor, low linolenic acid soybean line were therefore used for the re-assay. The amount of 1-octen-3-ol formed from dehulled seeds was about half that of intact seeds, confirming the hypothesis that components such as fungi and fungal enzymes in soybean hulls contribute to the formation of 1-octen-3-ol (Table 13). Lipoxygenase-free soybeans often produce higher levels of 1-octen-3-ol (Table 3). It is theorized that lipoxygenases may play a role in inhibiting the growth of mold, and thus in the absence of lipoxygenases, mold contamination will be greater, allowing more fungal enzymes and thus more 1-octen-3-ol to be formed. Lipoxygenase-containing low-odor soybeans identified by the screen of the present invention tend to have a lower 1-octen-3-ol range than higher odor soybeans (e.g., Table 12).

Table 12A 1-A4: color and odor generating properties of low linolenic soybean lines. Color value of L^*(brightness of light), a^*(Green-Red) and b^*(blue-yellow).

Table 12a1.
										Type of hybridization	Linolenic acid (%)	Hexanal (mu g/g)	Hexanol (μ g/g)	1-Octen-3-ol (μ g/g)	2, 4-decadienal (μ g/g)	12, 4-decadienal, hexanal, hexanol (. mu.g/g)	L*	a*	b*
J-2J-1J-3	4.03.73.5	5.07.37.6	0.50.60.7	4.45.16.3	4.04.88.6	9.612.716.8	79.982.681.9	1.50.41.0	27.527.026.8
										J-4J-5	4.64.2	5.86.5	5.15.1	4.34.7	9.48.9	20.320.5	81.981.8	0.90.7	27.526.3
F-2	2.7	14.8	0.4	3.9	7.6	22.7	82.6	0.6	26.2
										E-11	2.7	19.9	5.1	4.4	7.0	32.0
G-31	4.1	20.3	1..1	5.5	11.0	32.4
										I-7	3.1	16.0	5.1	3.5	12.2	33.3
J-12I-4	3.3	16.515.5	5.15.5	4.23.6	12.513.7	34.134.3
										G-21	4.1	20.7	2.1	6.0	12.1	34.8
F-20F-3E-13	4.92.83.0	24.623.323.4	1.40.95.1	3.89.14.1	9.311.77.4	35.335.935.9
										G-30F-6G-27	3.2	21.823.022.6	1.31.70.9	5.45.96.6	13.011.612.9	36.036.336.4
G-16	4.2	19.5	3.5	4.6	13.6	36.6
										G-10G-15	4.14.4	17.318.9	3.63.0	3.54.8	16.015.4	36.937.2
G-28	4.2	24.8	1.0	7.0	11.4	37.2
										H-1F-17	3.43.9	17.225.7	5.11.3	3.05.3	15.210.5	37.537.5
G-26	3.8	23.1	1.0	5.1	13.7	37.9
										E-14J-16	3.03.2	20.919.8	5.15.1	5.34.5	11.913.0	37.938.0
E-6	2.9	17.4	5.1	4.3	15.6	38.0
										E-3F-10	2.94.3	17.820.1	5.15.1	3.03.0	15.413.0	38.238.3
F-1	2.7	21.6	1.5	5.4	15.6	38.6

Table 12a2.
							Type of hybridization	Linolenic acid (%)	Hexanal (mu g/g)	Hexanal (mu g/g)	1-Octen-3-ol (μ g/g)	2, 4-decadienal (μ g/g)	2, 4-decadienal, hexanal, hexanol (. mu.g/g)
E-7E-5	3.02.7	20.120.2	5.15.1	4.04.2	13.513.7	38.738.9
							G-8	4.2	27.1	3.5	4.1	8.3	38.9
E-16F-19	3.13.6	22.226.2	2.35.1	3.83.3	14.67.9	39.139.1
							G-19	4.3	20.0	4.2	4.6	15.2	39.4
F-25F-21G-11	3.92.84.2	23.422.922.1	0.71.14.0	9.57.24.6	15.315.713.6	39.439.639.7
							J-17E-15E-4	3.03.02.8	22.819.521.1	5.15.15.1	4.95.03.2	12.415.814.5	40.240.440.6
J-14	3.2	20.6	5.1	4.3	15.2	40.9
							G-24J-13	4.03.2	22.920.9	1.95.1	7.04.1	16.115.0	40.941.0
E-10	2.8	22.9	5.1	4.0	13.1	41.1
							F-31J-34	3.53.2	26.826.2	0.05.1	9.63.5	14.310.1	41.241.3
G-25	3.5	23.6	2.5	7.2	15.3	41.4
							F-5J-30	2.73.2	25.726.5	1.35.1	9.64.8	14.510.0	41.541.5
H-7	3.2	22.2	5.1	4.2	14.6	41.9
							G-22E-11	4.02.9	22.627.6	1.55.1	7.54.1	17.99.4	42.042.1
J-19	3.0	22.2	5.1	3.8	14.9	42.2
							F-13G-23	4.03.2	30.423.6	1.22.2	4.36.2	10.816.6	42.342.4
E-9	2.9	25.2	5.1	3.4	12.3	42.6
							F-22E-8	3.42.9	25.325.2	1.05.1	8.74.1	16.412.7	42.742.9
E-1	2.8	21.9	5.1	2.6	16.6	43.6
							F-8F-32	3.72.8	26.025.6	5.10.9	3.19.1	12.617.4	43.643.8
F-23	3.6	28.6	0.9	6.5	14.8	44.3

Table 12a3.
							Type of hybridization	Linolenic acid (%)	Hexanal (mu g/g)	Hexanal (mu g/g)	1-Octen-3-ol (μ g/g)	2, 4-decadienal (μ g/g)	2, 4-decadienal, hexanal, hexanol (. mu.g/g)
J-6G-29	2.74.0	23.823.7	5.11.5	4.26.8	15.719.3	44.644.6
							F-7	4.1	25.4	5.1	3.8	14.1	44.6
I-12G-17	2.94.1	22.020.8	5.14.2	3.94.1	17.720.1	44.845.1
							F-4	2.8	24.0	5.1	3.6	16.2	45.3
H-6G-9I-11	3.14.53.1	24.723.921.2	5.13.85.1	3.53.83.6	15.717.819.2	45.445.545.5
							G-14I-13J-15	4.12.93.1	22.821.124.4	3.35.15.1	4.94.85.6	19.519.616.8	45.745.846.3
F-33	3.0	29.0	0.9	10.1	17.6	47.5
							E-2J-32	2.83.0	25.331.5	5.15.1	3.44.6	17.411.2	47847.8
E-18	3.0	28.8	2.9	3.2	16.2	48.0
							H-5E-17	3.03.2	30.029.0	5.13.2	3.32.5	13.016.3	48.048.6
I-6	2.9	30.8	5.1	4.1	12.8	48.7
							J-33G-2	2.92.9	28.626.8	5.11.5	2.66.5	15.220.7	48.949.0
G-12	4.1	23.1	4.9	4.5	21.4	49.4
							I-8G-5	2.82.9	26.531.1	5.13.4	5.78.1	18.015.2	49.549.7
G-6	3.2	31.7	1.6	8.9	16.7	50.1
							J-23F-35	2.92.9	30.028.8	5.11.2	6.96.5	15.020.4	50.150.4
I-5	2.8	30.4	5.1	3.1	15.0	50.6
							G-20J-29	4.03.0	23.831.3	5.05.1	5.34.5	22.014.6	50.851.0
G-13	4.4	35.8	3.8	4.8	11.5	51.0
							I-0H-2	3.02.8	32.734.0	5.15.1	4.13.2	13.312.6	51.151.7
G-18	3.9	27.6	4.4	4.5	19.9	51.9

Table 12a4.
							Type of hybridization	Linolenic acid (%)	Hexanal (mu g/g)	Hexanal (mu g/g)	1-Octen-3-ol (μ g/g)	2, 4-decadienal (μ g/g)	2, 4-decadienal, hexanal, hexanol (. mu.g/g)
H-3	3.0	28.6	5.1	3.3	18.5	52.2
							F-18	2.9	33.3	5.1	4.5	14.0	52.4
F-26	4.6	31.4	1.6	4.5	19.3	52.4
							G-4	3.1	28.7	2.4	7.6	21.4	52.5
J-9	3.5	29.9	5.1	5.4	17.7	52.7
							J-31	3.0	33.6	5.1	4.7	14.1	52.8
J-27	3.1	34.5	5.1	5.9	13.9	53.5
							G-7J-24	2.63.1	32.231.1	5.15.1	8.86.2	16.317.7	53.653.9
I-1	2.9	32.1	5.1	4.5	17.0	54.2
							I-2I-3	3.02.9	32.134.0	5.15.1	4.85.2	17.216.2	54.355.3
J-11	3.3	34.8	5.1	6.2	16.1	55.9
							G-1H-4	2.72.9	29.836.0	4.25.1	5.43.8	22.216.1	56.157.2
I-9	3.1	30.4	5.1	4.7	22.0	57.4
							F-14J-10	4.33.1	41.735.8	2.05.1	4.44.8	13.817.3	57.558.2
J-25	3.1	37.6	5.1	7.0	16.7	59.3
							J-28J-18	3.13.3	38.339.1	5.12.4	4.87.8	16.118.2	59.559.6
J-21	3.1	37.7	5.1	5.3	17.1	59.9
							J-22F-30	3.23.1	38.035.7	5.10.8	7.213.2	16.923.8	60.060.2
J-26	3.1	37.1	5.1	5.2	19.0	61.2
							J-8J-7	3.33.2	37.638.6	5.15.1	5.55.5	18.918.3	61.662.0
F-12	4.8	36.5	5.1	3.9	21.0	62.5
							G-3F-28F-24	2.92.94.4	32.643.542.0	2.52.43.5	9.86.26.4	27.716.918.3	62.762.863.7
F-34	3.0	35.3	1.5	7.8	27.0	63.8
							E-19F-29	3.03.6	40.942.0	4.63.5	1.74.6	19.320.8	64.866.2
E-12	2.8	39.9	5.1	4.4	21.8	66.7
							J-20F-27	2.92.9	41.542.1	5.10.9	5.38.1	21.526.5	68.169.6
F-11	3.6	41.3	5.1	3.5	23.7	70.1
							F-9F-16	4.64.1	46.742.8	1.45.1	5.24.5	21.922.5	70.170.4
F-15	3.7	54.5	2.2	6.7	25.8	82.5

Table 13: reduced 1-octen-3-ol formation in odor assays when soybeans are dehulled prior to being made into a soybean meal

Soybean 14	1-Octen-3-ol (μ g/. mu.g)
					Whole seed	Shelling	Hulling Stdev
J-2J-1	4.395.08	2.172.25	0.130
				J-3j-4	6.264.27	2.361.35	0
HiBC	10.36	3.19	0.09

Example 7

Effect of pH on odor formation

The standard odor assay provided by the present invention involves the addition of soy flour to water, resulting in odor formation at about ph 6.3. The purpose of the following experiment was to determine if species that produce low levels of odor under these conditions, also produce low levels of odor under other pH conditions. The odor-generating properties of the commercial control soybeans were compared with the low odor-generating line (A-4). Line A-4 produced lower levels of decadienal and hexanal at pH3.0 and pH5.5 and at pH7 and pH9.2 (Table 14). These data demonstrate that assays performed without buffer can be used as a method of selecting soybeans that can produce low levels of odor over a wide range of pH conditions. The highest concentrations of hexanal and 2, 4-decadienal were produced at pH9, at which 1-octen-3-ol was produced in the least amount (Table 14).

Table 14: effect of pH on odor generation of commercial control soybeans and low odor-producing soybeans (a-4 of figures 4 and 5 was harvested in 2003). The odor assay used for this measurement was the same as that of example 1, except that soy flour was added at 0.1M K at each pH (3.02, 5.45, 7.01, and 9.16)₂HPO₄In (1).

pH	Decadienal (μ g/g)				Hexanal (mu g/g)				1-Octen-3-ol (μ g/g)
													Control	Stdev	A-4	Stdev	Control	Stdev	A-4	Stdev	Control	Stdev	A-4	stdev
	3.02	3.60	0.98	1.79	0.96	18.47	4.40	7.35	0.76	13.37	7.86	11.71													2.79
5.457.01													8.038.10	1.971.90	3.653.51	2.270.64	36.4930.75	2.669.50	11.3010.68	1.780.74	12.4110.57	6.564.17	8.066.03	3.623.87
	9.20	37.72	33.17	9.12	3.66	60.38	30.20	27.93	6.11	2.10	1.71	2.98													1.67

Example 8

Effect of Soy protein composition on Soybean milk precipitate

This example illustrates that the level of sediment formed in soymilk made from soybeans of the invention having improved protein composition is lower. Protein-containing precipitates have a negative impact on the organoleptic quality of soymilk, since it is undesirable to have particles perceived in beverages.

Soybean milk preparation control low odor soybeans (A-4) and soybeans containing about 39% beta-conglycinin and about 13% glycinin were ground with Mega Grinder to make soybean meal. Each soy flour sample was added to 36.75 grams of water (4 ℃) in a 50mL disposable polypropylene centrifuge tube in an amount to give a final protein concentration in the mixture of 3.3% by weight and sonicated at a power output control set point of 8 for 15 seconds. The sonicated samples were centrifuged at 8,000rpm for 10 minutes at 4 ℃ using an Eppendorf Centrifuge5804R Centrifuge. The supernatant (soymilk) was decanted into a 50mL disposable polypropylene centrifuge tube. A portion (27.25 grams) of each soymilk was transferred to another 50mL disposable centrifuge tube. The above samples were prepared in duplicate to address the differences in sucrose addition as follows. Sucrose (0.6987 g) was added to 27.25 g of soymilk before or after the heat treatment.

A50 mL disposable polypropylene centrifuge tube was placed in a 95 ℃ silicone oil bath for 5 minutes to heat treat the soybean milk sample, which was then transferred to an ice bath to cool, followed by cryopreservation for 30 days. Over time, a precipitate formed in the sample. The formed precipitate was quantified as follows. The tube was tipped back and forth to disperse the sediment at the bottom of the tube. The soymilk sample was transferred to a weighed centrifuge tube and the final weight was recorded. The centrifuge tube was placed in an Eppendorf centrifuge5804R centrifuge and centrifuged at 8,000rpm for 2 minutes at 25 ℃. The soymilk supernatant was decanted and the weight of the remaining precipitate was calculated. The amount of sediment was recorded as a percentage of the weight of soymilk (% sediment-100 x sediment weight/soymilk weight).

The control soymilk had at least twice the amount of sediment as the high β -conglycinin soymilk (table 15).

Table 15: effect of soy protein composition on soymilk precipitate a-4 is control soybean. HBC is high beta-conglycinin, low glycinin soybean.

Type of soybean	Sucrose addition	% precipitate
			A-4	Before heating	1.8
A-4HBC	After heating and before heating	2.00.7
			HBC	After heating	1.0

The above examples show that even when soy contains lipoxygenases 1, 2 and 3, a unique composition with low odor generation levels is produced. It has also been demonstrated that it is possible to select soybeans with improved organoleptic properties, wherein the soybeans are selected according to: the amount of glycinin, free arginine and asparagine, yellow pigments and polyunsaturated fatty acids in the soybeans, and the amount of 2, 4-decadienal, hexanal, hexanol and 1-octen-3-ol odors produced by grinding aqueous soybean suspensions. The disclosed method is used to estimate the potential of soybeans to produce 2, 4-decadienal, hexanal, hexanol, and 1-octen-3-ol, among other more intense odors in soybean ingredients and foods. In this soy odor estimation method, the value of incubating soy flour in water at a 1: 4 ratio is to obtain the following unique observations: 2, 4-decadienal forms rapidly in suspension at normal temperature, making it possible to cultivate soybeans containing lipoxygenases 1, 2 and 3 with very low odor-generating levels.

In each example, it was also demonstrated that it was possible to produce an endogenous soy composition having greater than 30% beta-conglycinin and less than 25% glycinin, with normal or low free arginine and free asparagine. It has further been demonstrated that low odor and color characteristics can be combined with high beta-conglycinin compositional characteristics, and that combinations with low and medium oleic soybeans are further contemplated. Glycinin is a source of insoluble protein in soy ingredients and foods and can cause precipitates in beverages and unpleasant mouthfeel. Free arginine and asparagine can produce ammonia during processing, which further reacts with odorous compounds such as 2, 4-decadienal to form a strong odor such as 2-pentylpyridine. Linoleic and linolenic acids are polyunsaturated fatty acids, substrates for odor formation; reducing their content in soybeans will help to reduce the formation of odors. Soy coloring contributes to the undesirable color of the soy product, further affecting the sensory response. Collectively, soy compositions with high beta-conglycinin, low free arginine and asparagine, low odor and low color, and low polyunsaturated fatty acids are the most valuable compositions of the present invention for producing organoleptically pleasing soy protein ingredients and foods. It is also recognized that these compositions do not lack the health attributes associated with the soy protein component, since β -conglycinin is associated with the cholesterol and triglyceride lowering properties of soy protein (Duranti et al, 2004), and is involved in the inhibition of atherosclerosis (Adams et al, 2004).

Example 9

Additional precipitation analysis of Low-odor Soybean compositions

The study of example 8 was repeated with the following changes. A commercial soybean control (AG3302) was added. The soybeans are dehulled before milling, and the soybean meal is sieved before adding to water. The supernatant (soymilk) was transferred to a pre-weighed centrifuge tube. Sucrose was added before heat treatment to give a final sucrose concentration of 2.5% (w/w). The samples were stored in a refrigerator for 21 days. The height of the soybean milk precipitate in the centrifuge tube was measured, and then the sample was centrifuged at 8,000rpm for 5 minutes. The supernatant was decanted and weighed, the pH of the supernatant was determined (pH 6.7 for all samples), and the centrifuge tubes containing pellet (pellet) were weighed. The weight percentage of wet soy precipitate was calculated for each sample (weight% soy precipitate-100 x weight of wet soy precipitate/(weight of wet soy precipitate + weight of soy supernatant).

The beneficial effects of HBC and low odor soybeans in reducing protein precipitate formation were found with dehulled soybeans (table 16). The HBC soymilk reduced the sediment 2.2-fold compared to the low odor soybean line and 7-fold compared to the control soybean. The precipitate of low-odor soybeans was surprisingly found to be reduced by a factor of 3.1 compared to the control. It is likely that the low odor profile reduces precipitate formation by limiting free radical formation and protein oxidation. This therefore indicates more beneficial properties of an optimized composition combining the high β -conglycinin trait and the low odor trait.

Table 16: effect of high beta-conglycinin character and Low odor character on Soybean milk sediment height and sediment-pellet weight compared to control Soybean composition

	Height of precipitate (mm)	Weight of granular precipitate (%)	Weight of granular precipitate Stdev
				HBC	0	2.1	0.14
A-4	8	4.7	0.42
				Control	14	14.8	2.8

Example 9

Preparation of dehulled soybean meal and soy protein isolate

Control soybeans (AG3302) and soybeans having greater than 30% total protein as beta-conglycinin and less than 25% total protein as glycinin and containing less than 2500 micrograms/g free arginine plus asparagine (DJB2104GOR, EXP319AP) were dehulled to produce dehulled soybean meal which was then further processed to produce a soy protein isolate component according to the following procedure.

Table 17: composition of beta-conglycinin and glycinin in different soybean lines

Soybean line	B-conglycinin (% of total protein)	Glycinin (in% of total protein)	Free Arg (ug/g)	Free Asn (ug/g)
					AG3302	24.1	33.6	1455	155
DJB1804BOR	33.9	12.9	1230	93
					EXP319AP	40.1	12.5	2197	224

1. Soybeans were adjusted to about 10% moisture and tempered at room temperature (temper).

2. Crushing the soybeans with a crusher.

3. The crushed soybeans are dehulled using an aspirator.

4. The crushed and dehulled soybeans are softened with a cooker at 50-60 ℃.

5. The flaking machine flaked the softened soybeans.

6. Extracting the soy flakes with hexane.

7. And removing the solvent from the defatted soybean meal to obtain the soybean flakes.

8. Grinding soybean slices to obtain soybean powder.

9. Water was added to a 300 liter jacketed tank, brought to 50 ℃ with 40% NaOH and pH 9.0. Defatted soy flour was added and mixed. The ratio of water to soy flour was 12/1 (w/w). The extraction time was 45 minutes.

10. The dissolved soy protein is recovered from the extraction slurry using an automatic cleaning disk centrifuge (back pressure 58-60 psi).

11. The clear protein solution was adjusted to pH4.5 by the addition of hydrochloric acid (18%) and allowed to react at 45 ℃ for 30 minutes.

12. The precipitated protein was recovered by an automatic cleaning disk centrifuge.

13. The protein clot was washed twice with acidified water (pH4.5+/-0.1, 30-35 ℃). The ratio of wash water to compacted wet solids was 6: 1 (w/w). The protein clot was recovered after each wash using an automatic wash dish centrifuge (back pressure 58-60 psi).

14. The washed clot was mixed with sodium hydroxide (30%), the pH adjusted to 6.8 with 30% NaOH, and then heat treated at 116 ℃ for 7.5 seconds. The pH was then adjusted to 6.8.

15. The protein solution was adjusted to 45-55 ℃ and spray dried with an inlet air temperature of 204-215 ℃ and an outlet air temperature of 82-88 ℃.

16. The nitrogen solubility index of the soy protein isolate component is determined. A portion of the sample was suspended with stirring in water at 30 ℃ for 2 hours and then diluted to a known volume with water. A portion of the sample extract was centrifuged and an aliquot was taken for protein analysis. Another portion of the sample was analyzed for total protein in the same manner. Water soluble protein is calculated as a percentage of total protein, proportional to water soluble nitrogen as a percentage of total nitrogen.

As a result:

the nitrogen solubility index of the soy protein flour (table 18) is proportional to the amount of β -conglycinin in the soy used to prepare the soy protein component: NSI 1.4716 (% β -conglycinin) + 7.4502; r square 0.9975. A decrease in the level of soluble protein can improve the organoleptic quality (e.g., a smoother, fresher mouthfeel) of food products formulated with soy protein.

Table 18: nitrogen solubility index

Soybean line for preparing soybean protein isolate	NSI(％)	Standard deviation of
			AG3302	42.7	2.5
DJB1804BOR	58.0	2.5
			EXP319AP	66.0	2.3

The soy protein isolate produced from each soybean was similar in amino acid composition, except that the high β -conglycinin (HBC) fraction had a lysine content of about 6% higher than the control.

Table 19: total amino acid composition of soy protein isolate

	AG3302	DJB1804BOR	EXP319AP
				Aspartic acid	114.5	111.2	114.7
Threonine	37.6	37.3	36.8
				Serine	50.8	52.0	52.7
Glutamic acid	203.3	192.0	202.2
				Proline	55.5	53.8	55.2
Glycine	41.6	40.0	39.8
				Alanine	42.9	42.2	41.9
Valine	49.1	49.6	49.7
				Methionine	13.3	13.1	12.3
Isoleucine	44.3	44.5	44.9
				Leucine	81.1	81.3	82.6
Tyrosine	38.4	39.1	39.3
				Phenylalanine	50.9	51.8	53.7
Histidine	23.7	25.9	26.3
				Lysine	62.3	65.4	66.5
Arginine	73.8	71.9	75.8
				Cysteine (after oxidation)	13.1	14.6	13.6
Methionine (after oxidation)	14.7	14.5	13.3
				Tryptophan	12.1	12.0	11.4

Example 10

Preparation of fermented soybean product from dehulled soybean meal

Dehulled soybean meal was prepared from commercial soybean (AG3302), high β -conglycinin soybean (DJB2104GOR), and low odor producing soybean lines (03JBK8-25), all of which were harvested in 2004 in the united states.

Method and detection method for the preparation of a fermented product from soybeans:

1. crushing the soybeans with a crusher.

2. The crushed soybeans are dehulled using an aspirator.

3. The soybean pulp (dehulled soybean) was ground by passing through a hammer mill 1 time and a pin mill 5 times.

4. Packaging the hulled soybean powder with plastic bag, and placing into a fiberboard bucket.

5. The protein content of the soy flour was determined.

6. Dehulled soybean meal (3 ℃) was added to water (3 ℃) to a protein content of 3.5% by weight and mixed with a hand-held homogenizer for about 1-2 minutes.

7. The soy flour suspension was warmed in a plate heat exchanger and then the suspension was treated with steam at 141 ℃ for 3.5 seconds followed by degassing and cooling to about 4 ℃.

8. Filtering the heat-treated suspension to remove fiber to obtain soybean milk.

9. Milk flavor, sugar (3%) and salt (0.2%) were added to the soybean milk, and mixed with a hand-held homogenizer.

10. The seasoned soybean milk was warmed in a plate heat exchanger, and then the suspension was treated with steam at 141 ℃ for 3.5 seconds, followed by degassing, cooling to about 4 ℃, and packaging in a sterile container.

11. Samples of soymilk, each containing 2.2% protein, were weighed into sterilized quart jars (quart jar) and warmed in a microwave for 45-60 seconds (about 24 ℃).

12. Sugar (3.1%), vanilla (0.4%) and fermented soy yogurt (l.bulgaricus, s.thermophilus, l.acidophilus, b.bifidum, l.casei, l.rhamnosus) (8%) were added to the soy milk and the samples were mixed.

13. The soymilk culture contained was incubated in a 43 ℃ incubator for 4 hours and then removed for freezing overnight (4 ℃).

14. pH and viscosity measurements were performed on the frozen samples and sensory assessed by a 3-person taste panel without knowledge of the sample formulation. The viscosity was measured with a Brookfield viscometer with Spindle #3 at 20 rpm.

As a result: the fermented product obtained from DJB2104GOR has a flavor profile (flavour) which is pleasing and should be suitable for making fruit flavored smoothies. The product made from 03JBK8-25 has a pleasant flavour profile and should be well suited for the manufacture of sour cream or dipped goods (dip products). It is theorized that the combination of the high β -conglycinin trait and the low odor producing trait should also produce a pleasing fermented soy milk product. The lower viscosity of the high β -conglycinin protein material may help produce a higher protein product at the same thickness level.

Table 20: soybean composition and odor characteristics

Soybean	B-conglycinin (% of total protein)	Glycinin (in% of total protein)	Oil (Dry matter)	Protein (Dry matter)	Free Arg (ug/g)	Free Asn (ug/g)	Hexanal (ug/g)	Hexanol (ug/g)	1-Octen-3-ol (ug/g)	2, 4-decadienal (ug/g)
											DJB2104GOR	37.7	14.7	18.9	39.5	2197	142	8.8	3.17	5.9	11.7
AG3302	24.1	33.6	22.4	38.1	1455	155	12.5	4.47	3.0	13.7
											03JBK8-25	25.7	34.3	19.1	38.2	1711	116	2.6	1.39	3.3	5.6

Table 21: flavor profile of fermented products

	Initial pH	Initial pH	Change in pH	Viscosity of the oil	Sensory evaluation
						Identity of sample	(4℃)	(4℃)		(centipoise)	Comments
						AG3302	6.828	4.629	2.199	2025	Has the consistency of smoothie, has a small amount of clotted small pieces, soil smell, fermentation flavor, sourness, vanilla flavor, cooked feeling, and is not as sweet as other samples.
DJB2104GOR	6.922	4.581	2.341	1095	Is thinner, sweeter, "brighter", and more stable than other samples, and gives off vanilla flavor, acid.
						03JBK8-25	6.835	4.662	2.173	1948	The rice pudding-like texture, flavor like sour cream, was the thickest sample, and the sour profile appeared to be a different type, quite mild, with some dry mouthfeel than the other samples.

Example 11

Demonstration of a combination of Low odor and high beta-conglycinin

This example illustrates the combination of low odor generating properties with a high beta-conglycinin composition. It was demonstrated that a low odor soy composition with reduced glycinin content and increased beta-conglycinin content could be produced. Hybrid types such as A and E (Table 3) were combined with high β -conglycinin germplasm with the A3233/B2G2/A1923 lineage.

Protein analysis was performed as follows: eight soybean seeds were pooled and ground with CAT Mega-Grinder (SOP Asci-01-0002). The ground samples were stored at 4 ℃. For analysis, -30mg of powder for each sample was weighed into one well of a 96 well 2ml microtiter plate. The protein was extracted with shaking in 1.0mL of Laemmlis SDS buffer pH6.8 containing 0.1M Dithiothreitol (DTT) as a reducing agent for 1 hour. After centrifugation, a portion of each extract was further diluted in SDS buffer to give 0.2-0.5. mu.g/. mu.L of total protein, and then heated to 90-100 ℃ for 1 minute, followed by cooling. For each sample, 1-2 μ g of total protein was loaded onto 26 lanes of 15% T-gradient Tris/HCl Criterion gel using a 12-channel pipette. Molecular weight standards and parental controls were added to two lanes of each gel. The gel was electrophoresed until the tracer dye reached the bottom of the gel (. about.1.2 hours), then stained overnight in colloidal Coomassie blue G-250, destained in deionized water, and developed using a GS 800Calibrate densitometer. Quantification was performed using Bio-Rad Quantity one (TM) Software. The software was used to determine the relative amount of each band in the sample lane. The bands for% glycinin subunit and% β -conglycinin subunit are reported as relative percentages of total protein in the lanes. The a 5-glycinin subunit was not quantified and was not included in the total acidic glycinin values. Sample identity and weight were tracked using Master LIMSTM.

The low odor producing soybean with the high β -conglycinin trait was demonstrated in comparison to some lines that did not have the low odor trait (table 22). The first line in the table was subjected to repeated odor analysis. It is shown that several strains lack glycinin and that the resulting composition after oxidation under mild aqueous conditions has less than 20 μ g/g total 2, 4 decadienal plus hexanal per gram of ground seeds. Further evaluations were also made for other characteristics of the resulting compositions (e.g., yield, free amino acid color and fatty acid composition).

Table 22: high beta-conglycinin soy composition odor generating properties and protein subunit composition.

Sample ID	Hexanal (ug/g)	Hexanol (ug/g)	Hexanal + hexanol (ug/g)	1-Octen-3-ol (ug/g)	2, 4-decadienal (ug/g)	Alpha′BC(％)	AlphaBC(％)	BetaBC(％)	AcGly(％)
										JB0305620.0102.0001	ND	2.5	2.5	6.9	3.8	16.0	24.4	8.1	0.0
JB0305620.0102.0001	4.4	2.5	7.0	8.2	4.0	16.0	24.4	8.1	0.0
										JB0305616.0349.0016	5.7	2.3	8.0	8.9	3.0	14.7	18.3	6.3	12.1
JB0305597.0181.0008	7.1	2.2	9.4	5.4	3.0	14.0	15.2	4.4	5.0
										JB0305617.0178.0015	5.3	2.0	7.3	1.5	5.2	15.4	17.4	3.5	0.0
JB0305618.0035.0025	7.2	1.7	8.9	8.9	3.8	16.4	19.2	6.6	0.0
										JB0305616.0349.0011	7.1	2.2	9.3	10.9	3.8	13.6	18.8	8.7	2.6
JB0305611.0213.0020	5.9	2.7	8.6	9.9	4.5	15.3	18.5	5.7	1.2
										JB0305620.0102.0018	7.8	2.6	10.4	5.5	3.0	16.8	23.9	10.1	0.0
JB0305620.0102.0004	7.9	2.3	10.2	9.7	3.3	16.0	23.0	9.0	0.0
										JB0305617.0094.0031	7.3	2.3	9.5	1.6	4.6	16.8	21.4	6.6	3.2
JB0305617.0178.0010	8.0	1.9	9.9	12.1	4.4	14.8	16.3	3.4	0.0
										JB0305611.0098.0009	7.7	2.3	10.0	8.8	4.4	14.9	18.2	10.3	6.7
JB0305611.0213.0004	7.3	2.2	9.5	12.7	5.0	14.0	16.1	4.1	0.9
										JB0305611.0380.0010	7.5	2.2	9.7	6.7	4.9	16.3	19.5	6.5	3.8
JB0305611.0213.0022	7.0	2.4	9.4	11.6	5.3	13.6	15.4	6.0	1.6
										JB0305619.0275.0007	16.8	4.5	21.3	14.0	14.0	14.5	13.4	3.9	0.0
JB0305620.0078.0012	19.4	9.2	28.6	4.5	7.5	17.0	18.7	7.7	0.0
										JB0305618.0035.0021	21.1	5.1	26.2	7.0	10.2	11.6	13.5	3.4	0.0
JB0305618.0047.0021	20.5	4.2	24.7	16.5	12.2	12.5	13.5	2.3	0.0
										JB0305620.0282.0008	24.7	3.9	28.6	7.4	8.4	12.3	13.9	3.3	0.0

No detection of Gly ═ acidic glycinin, ND ═

Preservation information

Monsanto Technology LLC soybean 0119149 seeds, disclosed hereinabove and described in the claims, have been deposited under the Budapest treaty at the American type culture Collection (ATCC, 10801 University Boulevard, Manassas, Va.20110). The deposited line 0119149, used herein in the various working examples and tables, is also identified as "a-4" and "03 JBK 8-25". The deposit has ATCC accession number PTA-6197 and a preservation date of 2004, 9 months and 10 days. The deposit will remain in the holding mechanism for a period of 30 years, or for a period of 5 years after the last request, or until the expiration of the life of the invention, depending on which period is longer, and will be replaced as needed during the holding period.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps and in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain substances which are both chemically and physiologically related may be substituted for the substances described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims (36)

1. A soybean pulp composition produced from soybeans comprising lipoxygenases 1, 2 and 3, said composition comprising more than 10% linoleic acid as a percentage of total fatty acids, and less than 20 μ g of total 2, 4-decadienal plus hexanal plus hexanol per gram after oxidation under mild aqueous conditions.

2. The composition of claim 1, comprising less than 4% linolenic acid as a percentage of total fatty acids.

3. The composition of claim 1, comprising less than 2000 μ g/gram of free arginine and less than 400 μ g/gram of free asparagine.

4. The composition of claim 1, said composition having a color according to wherein L^*Represents lightness and b^*CIE-L representing the hue on the blue (-) -yellow (+) color axis^*a^*b^*The system monitors and measures b^*A value less than 30 and L^*The value is greater than 80.

5. The composition of claim 1 comprising a 1-octen-3-ol content of less than 4 μ g per gram after oxidation under mild aqueous conditions.

6. The composition of claim 1 having greater than 30% of the protein as β -conglycinin.

7. The composition of claim 1 having less than 25% of the protein as glycinin.

8. The composition of claim 1, having a linoleic acid concentration of between 10% and 60% of the total fatty acids.

9. A soybean pulp composition having greater than 30% of the protein as β -conglycinin and less than 25% of the protein as glycinin, and comprising less than 5,000 μ g/g free arginine and less than 900 μ g/g free asparagine.

10. The composition of claim 9, comprising less than 2,000 μ g/gram of free arginine and less than 400 μ g/gram of free asparagine.

11. The composition of claim 9 comprising a 1-octen-3-ol content of less than 4 μ g/g after oxidation under mild aqueous conditions.

12. The composition of claim 9 comprising less than 20 μ g/g total 2, 4 decadienal plus hexanal plus hexanol after oxidation under mild aqueous conditions.

13. The composition of claim 9, having a linolenic acid concentration of between 1.5% and 14% of total fatty acids.

14. The composition of claim 9 having a linoleic acid concentration of between 10% and 60% of the total fatty acids.

15. The composition of claim 9, defined as lacking lipoxygenase-2.

16. The composition of claim 9, defined as lacking lipoxygenase.

17. The composition of claim 9, said composition being color-characterized in accordance with wherein L^*Represents lightness and b^*CIE-L representing the hue on the blue (-) -yellow (+) color axis^*a^*b^*The system is monitored, b^*A value less than 30 and L^*The value is greater than 80.

18. The composition of claim 9, comprising 67-69mg lysine per gram protein.

19. The composition of claim 9 comprising 72-80mg arginine per gram protein.

20. The composition of claim 9, comprising 28-30mg histidine per gram protein.

21. A soybean pulp composition having greater than 30% of the protein as β -conglycinin and less than 25% of the protein as glycinin, and comprising less than 20 μ g of total 2, 4-decadienal plus hexanal plus hexanol per gram after oxidation under mild aqueous conditions.

22. The composition of claim 21, defined as lacking lipoxygenase-2.

23. The composition of claim 21, having a linolenic acid concentration of between 1.5% and 14% of total fatty acids.

24. The composition of claim 21, having a linoleic acid concentration of between 10% and 60% of the total fatty acids.

25. The composition of claim 21, defined as lacking lipoxygenase-2.

26. The composition of claim 21, said composition being color-characterized in accordance with wherein L^*Represents lightness and b^*CIE-L representing the hue on the blue (-) -yellow (+) color axis^*a^*b^*The system is monitored, b^*A value less than 30 and L^*The value is greater than 80.

27. A method of analyzing the odor-generating characteristics of soybeans, said method comprising determining the level of at least one compound selected from the group consisting of 2, 4-decadienal, hexanol, hexanal, and 1-octen-3-ol.

28. The method of claim 27, wherein said determining the level of the compound comprises incubating a mixture of about 1 part soybean seed meal and about 4 parts water for a time of about 1 to about 40 minutes, and quantifying the amount of at least one compound selected from the group consisting of 2, 4-decadienal, hexanol, hexanal, and 1-octen-3-ol, and combinations thereof, with a deuterated standard of hexanal, hexanol, and decadienal.

29. The method of claim 28, wherein the soybean seed meal is prepared from dehulled soybeans.

30. A method of obtaining a soybean variety for producing soybeans with reduced odor-generating characteristics, the method comprising measuring the level of at least one compound selected from the group consisting of 2, 4-decadienal, hexanol, hexanal, and 1-octen-3-ol, and any combination thereof, in one or more soybeans from first and second soybean varieties, and selecting a variety that produces a seed with a lower level of said compound.

31. The method of claim 30, further comprising crossing a plant of the selected variety with another plant to produce a progeny, and measuring the level of at least one compound selected from the group consisting of 2, 4-decadienal, hexanol, hexanal, and 1-octen-3-ol, and any combination thereof, in one or more soybeans from said progeny.

32. A method of selecting a soybean variety that is resistant to fungal infection, the method comprising selecting a variety comprising less than 5 μ g 1-octen-3-ol per gram of seed as determined by incubating a mixture of about 1 part soybean seed meal and about 4 parts water for a time period of about 1 to about 40 minutes and measuring 1-octen-3-ol.

33. Seed of soybean plant of accession number 0119149, representative seed of said plant having been deposited with the ATCC, accession number PTA-6197.

34. A soybean plant 0119149 or parts, produced by growing the seed of claim 33.

35. The soybean plant of claim 34, said plant further defined as comprising a transgene.

36. A method of producing a soybean plant derived from the soybean plant 0119149, the method comprising the steps of:

(a) preparing a progeny plant derived from soybean plant 0119149 by crossing a plant of soybean plant 0119149 with another soybean plant, wherein a seed sample of soybean plant 0119149 has been deposited with the ATCC under accession No. PTA-6197;

(b) crossing the progeny plant to itself or to a second plant to produce seed for a next generation progeny plant;

(c) growing a next generation progeny plant from the seed and crossing the next generation progeny plant to itself or to a second plant; and

(d) repeating steps (b) and (c) for 3-10 additional generations to produce an inbred soybean plant derived from soybean plant 0119149.